FOR TREATING VIRAL RESERVOIRS

RELATED APPLICATION DATA The present application claims priority to U.S. Provisional Patent Application No.

61/896, 939, entitled "Combination Vaccination/ Antiretroviral Therapy for Treating Viral Reservoirs," filed on October 29, 2013, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions and methods useful for reducing viral reservoirs in a subject in need thereof. In certain embodiments, the present invention relates to compositions and methods useful for reducing reservoirs of HIV in a subject in need thereof.

BACKGROUND OF THE INVENTION

A viral reservoir is a cell or anatomical site in which a replication-competent form of a virus accumulates and persists with more stable kinetic properties than the main pool of actively replicating virus. Viral reservoirs represent a challenge to eradication of viral infection, even when viremia can be suppressed and maintained below detectable levels by means of anti-viral therapy.

It has become clear that viral reservoirs of HIV established early in infection represent a significant challenge to eradication of HIV infection. Potential reservoirs for HIV include resting memory CD4+ T cells, in which latent HIV shows minimal decay even in patients on highly active antiretroviral therapy (HAART). The persistence of virus in this reservoir is consistent with the biology of these cells and the long-term persistence of immunologic memory. While other HIV reservoirs have been reported, the reservoir in resting CD4+ T cells appears to be sufficient for a persistence of HIV infection in the majority of patients on HAART regimens.

New approaches are needed for reducing viral reservoirs and more particularly, for reducing HIV reservoirs, to eradicate or manage infection. SUMMARY OF THE INVENTION

Compositions and methods are provided for reducing viral reservoirs. Methods are provided for reducing viral reservoirs in a person on continuous antiretroviral drugs involving administering a stimulating ( "shock") agent to a subject in need thereof, followed by

administering an eradication ("kill") agent to reduce viral reservoirs. In certain embodiments, viral reservoirs are eradicated. In a preferred embodiment, the viral reservoir is an HIV reservoir.

In a first aspect, the present invention is a composition comprising one or more shock agents selected from a DNA plasmid, an HIV DNA vaccine, a CpG oligodeoxynucleotide, a TLR9 receptor agonist, an HDAC inhibitor, an NFkappaB activator, a cytosine methylation inhibitor, or an acetaldehyde dehydrogenase inhibitor.

In one particular embodiment, the shock agent is an HIV DNA vaccine.

In one embodiment, the shock agent is an HIV DNA vaccine comprising one or more safety mutations.

In one embodiment, the HIV DNA vaccine comprises (i) a prokaryotic origin of replication and (ii) a eukaryotic transcription cassette comprising a vaccine insert encoding

(a) a Gag protein comprising one or more safety mutations to inactivate zinc fingers;

(b) a Pol protein comprising one or more safety mutations to (i) inhibit integrase activity and (ii) inhibit polymerase, strand transfer, and/or RNase H activity of reverse transcriptase; and

(c) Env, Tat, Rev, and Vpu, with or without mutations. In one embodiment, the Gag protein has safety mutations that inactivate one or more of the zinc fingers corresponding to clade B C392S, C395S, C413S, and C416S.

In one embodiment, the Pol protein has safety mutations that delete the integrase sequence.

In one embodiment, the Pol protein has safety mutations that inactivate reverse transcriptase corresponding to clade B D185N, W266T, and E478Q. In one embodiment, the Pol protein has safety mutations to inhibit protease activity that corresponds to clade B D25A.

In one embodiment, the clade B HIV is strain HXB2.

In one embodiment, the HIV DNA vaccine has (i) a prokaryotic origin of replication and (ii) a eukaryotic transcription cassette comprising a vaccine insert encoding

(a) a HIV-1 clade B Gag protein comprising amino acid changes C392S, C395S, C413S and C416S to inactivate both zinc fingers; and

(b) a HIV-1 clade B Pol protein comprising:

(i) one or more safety mutations to delete integrase,

(ii) amino acid changes D185N, W266T and E478Q to inactivate reverse transcriptase activity, and

(iii) amino acid change D25 A to inhibit protease activity.

In one embodiment, the HIV DNA vaccine comprises a sequence encoding human GM-

CSF.

In one embodiment, the HIV DNA vaccine co-expresses GM-CSF.

In one embodiment, the shock agent comprises a CpG oligodeoxynucleotide (CpG

ODN).

In one embodiment, the shock agent comprises CpG ODN, wherein the CpG ODN is CpG-7909.

In one embodiment, the composition comprises two shock agents, wherein the shock agents are co-administered HIV DNA vaccine and CpG ODN.

In one embodiment, the shock agent is an HDAC inhibitor and more particularly, the shock agent is romidepsin.

In one embodiment, the shock agent is an acetaldehyde dehydrogenase inhibitor, and more particularly, the shock agent is disulfiram.

In another embodiment, the shock agent is a cytosine methylation inhibitor, and more particularly, 5-azacytidine. In a second aspect, the present invention comprises one or more kill agents selected from an HIV vaccine, a monoclonal antibody or combinations thereof.

In one embodiment, the HIV vaccine is selected from an MVA vaccine, an adenoviral vaccine, or a vesicular stomatitis virus (VSV) vaccine. In one embodiment, the adenoviral vaccine is a human chimpanzee adenoviral vector vaccine.

In one particular embodiment, the HIV vaccine is an MVA HIV vaccine.

In one particular embodiment, the kill agent is an MVA HIV vaccine including (i) a sequence encoding an HIV Env antigen inserted into deletion site II of an MVA, wherein the HIV Env antigen comprises gpl20 and the membrane spanning and ectodomain of gp41, but lacks part of all of the cytoplasmic domain, and (ii) a sequence encoding HIV Gag and Pol antigens inserted into deletion site III of an MVA, wherein the sequence encoding the HIV Gag and Pol antigens comprises safety mutations to limit packaging of viral R A and inactivate reverse transcriptase and delete the integrase coding sequence; and wherein the sequences of (i) and (ii) are under the control of promoters compatible with poxvirus expression systems.

In one embodiment, the safety mutations that inactivate reverse transcriptase and delete the integrase coding sequence correspond to clade B HIV D185N, W266T, and E478Q.

In one embodiment, the clade B HIV is strain HXB2.

In one embodiment, the kill agent is an antibody. In a particular embodiment, the kill agent is a monoclonal antibody or a recombinant Ab.. In one embodiment, the kill agent is a neutralizing antibody or a non-neutralizing antibody.

In one embodiment, the kill agent is a Ab that also initiates innate immune responses.

In one embodiment, the kill agent is a neutralizing Ab selected from antibodies such as 10-1074, 10E8, 17B, 2F5, 2G12, 447-52D, 448-D, 4E10, 670D, bl2, BB34, CAP206-CH12, CHOI, CH02, CH03, CH04, CH08, CH30, CH31, CH32, CH33, CH34, HJ16, M66.6, NIH45, NIH46, PG16, PG9, PGT121, PGT128, VRCOl, VRC02, VRC03, or VRC07.

In one embodiment, the kill agent is a non-neutralizing antibody selected from antibodies such as 120-16, 126-50, 246-D, 4B3, 50-69, 98-43, 98-6, b6, 3D6, A32, or F240. In a third aspect, the present invention is a method of reducing viral reservoirs in a subject on antiretroviral treatment in need thereof by: (i) administering one or more shock agents; and (ii) administering one or more kill agents, thereby reducing viral reservoirs.

In one embodiment, the kill agent is administered between about 1 and about 4 weeks, about 1 and about 2 weeks, or about 1 week after administration of the shock agent.

In one embodiment, the kill agent is administered between about 1 and about 10 days, about 1 and about 7 days, about 1 and about 5 days, about 1 and about 3 days, about 3 and about 7 days, about 5 and about 6 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after the shock agent.

In another embodiment, the kill agent is administered within 24 hours, i.e., within one day of the shock agent.

The method may comprise one or more additional steps. In one embodiment, the method further comprises the step of: (iii) measuring the presence of viral reservoirs for reduction. In one embodiment, viral reservoirs are measured at about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 weeks after step (ii). In another embodiment, the viral reservoirs are measured about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or 12 months after step (ii). In a further embodiment, the viral reservoirs are measured about 1, about 2, about 3, about 4, or about 5 years after step (ii). In another embodiment, the method further comprises determining the measuring the temporal levels of reactivated viral R A between step (i) and (ii) in order to determine peak levels of reactivated virus.

In another embodiment, the kill agent is administered at or about the time of peak virus reactivation. In one embodiment, the subject is undergoing antiretroviral therapy prior to, during, and/or after administration of the shock and/or kill agents.

In exemplary embodiments, the method is repeated two or more times. In a particular embodiment, the method is repeated two or more times at regular intervals. Where the method is repeated, the term "cycle" is used to refer to steps (i) and (ii), collectively. In one embodiment, the method is repeated at least 2 times, at least 3 times, at least 4 times, at least 5 times, or more than 5 times.

In one embodiment, the method is repeated and about the interval between cycles is about 1 to about 4 weeks, about 2 to about 4 week, about 3 about 4 weeks, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks.

In one embodiment, the method is repeated between about 2 and about 8 times, about 4 and about 8 times, about 6 and about 8 times, about 2, about 4, about 6 or about 8 times.

In another embodiment, the method is repeated more than 8 times.

In another embodiment, the time between step (i) and (ii) is substantially the same for all shock/kill cycles.

In one embodiment, the method is repeated until reservoirs are undetectable by reservoir assay.

In one embodiment, the method is repeated until viral reservoirs are reduced.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides graphs showing that CpG ODN, but not GpC ODN or GEO-D03 DNA, induce HIV-1 activation in ACH-2 cells. In FIG. 1A, cells were seeded at lxlO ⁶ cells/well. In FIG. IB, cells were seeded at 0.3xl0 ⁶ cells/well. Expression of HIV-Gag was determined by intracellular staining using mouse mAb KC57 (Beckman Coulter) and flow cytometry.

Stimulation with phorbol-12-myristate- 13 -acetate and ionomycin achieved 49% activation of HIV (not shown).

FIG. 2 provides graphs showing viral loads (in copies/mL plasma) in patients

participating in the GV-TH-01 clinical trial. "D" indicates vaccination with the GEO-D02 DNA vaccine. Of the nine HIV-infected, ART-suppressed participants who received the GEO-D02 and MVA62B vaccines in the trial, four (Participants 01-5, 01-6, 01-8 and 01-9 above) showed transient increases in viral load that occurred one week after receipt of the GEO-D02 DNA vaccine. Transient reactivations of latent virus that appeared to be stimulated by DNA

inoculations are indicated with arrows, and the corresponding participant IDs are circled.

FIG. 3 provides graphs showing reservoir measurement for all participants. Integrated

DNA results are shown in A, and inducible RNA (TILDA) results are given in B. Participant 01- 7 (indicated by shaded box), whose viral R A transiently increased at the time of the second MVA vaccination, experienced marked reductions in integrated DNA and inducible RNA.

*Pre-Vaccine indicates the time point prior to the first DNA vaccination for all participants other than 01-7. Because this sample was not available for participant 01-7, the 01-7 Pre- Vaccine time point is from Week 10, between the second DNA vaccination and the first MVA vaccination.

FIG. 4 provides a schematic of an exemplary shock/kill administration regimen for the flushing and killing of cells expressing reactivated HIV to reduce viral reservoirs .

DETAILED DESCRIPTION OF THE INVENTION Compositions and methods are provided for reducing viral reservoirs in a subject in need thereof. As used herein, the term "reducing viral reservoirs" refers to reduction of viral reservoirs compared to the levels of viral reservoirs present before treatment, including total elimination or eradication of viral reservoirs, i.e., reduction to zero or undetectable levels. Other similar terms include "flushing", "clearing" or "eradicating" viral reservoirs. Methods are provided for reducing viral reservoirs in a subject in need thereof, comprising (i) administering one or more stimulating or "shock" agents to stimulate the viral reservoir; and (ii) administering one or more eradication or "kill" agents, thereby reducing viral reservoirs. The viral reservoir may be, for example, an HIV viral reservoir.. These methods provide a two-step approach for flushing or clearing viral reservoirs in order to eradicate infection/ preventing further infection. To prevent spread of virus expressed by reactivated cells, (i) and (ii) are conducted in patients maintained on antiretroviral therapy.

Slight increases in viral levels have been surprisingly observed where combination DNA/MVA vaccines are administered in the presence of anti-retroviral therapy (ART). While not to be bound by any particular mechanism, it is believed that the HIV DNA vaccine acts as a shock agent and stimulates the reservoirs to express virus, making the infected cells susceptible to killing by the CD8+ T cells elicited by the MVA vaccine, which acts as an eradication or kill agent. The ART drugs reduce or prevent virus spread to uninfected cells

Other shock and kill agents are useful in this two-step approach to reducing viral reservoirs in a subject in need thereof. The virus may be HIV or another viral agent. I. Shock agents

As used herein, the term "shock agent" refers to a compound capable of stimulating viral reservoirs so as to activate or increase expression of latent virus, resulting in transiently increased viral load. The shock agent may be, without limitation, a small molecule shock agent or a nucleic acid shock agent such as an oligonucleotide, a nucleic acid vaccine or the like.

Methods of measuring viral load are known to those of skill in the art.

A. Nucleic Acid Vaccines

Accordingly, the present methods employ shock agents comprising nucleic acid sequences that induce an immune response to a virus (e.g., HIV-1), including nucleic acid analogs of those sequences and compositions containing those nucleic acids (whether vector plus insert or insert only or vector only; e.g., physiologically acceptable solutions, which may include carriers such as liposomes, calcium, particles (e.g., gold beads) or other reagents used to deliver DNA to cells).

The advantage of using nucleic acid compositions as compared to other shock agents described herein is that nucleic acid vaccines have improved an side effect profile. Certain shock agents described herein cause substantial systemic and local adverse events. However, the nucleic acid vaccines described herein activate viral reservoirs in the presence of very limited local or systemic reactogenicity.

The analogs can be sequences that are not identical to those disclosed herein, but that include the same or similar mutations (e.g., the same point mutation or a similar point mutation) at positions analogous to those included in the present sequences. For example, a given residue or domain can be identified in various HIV clades even though it does not appear at precisely the same numerical position. The analogs can also be sequences that include mutations that, while distinct from those described herein, similarly inactivate a viral gene product (e.g., an HIV gene product). For example, a gene that is truncated to a greater or lesser extent than one of the genes described here, but that is similarly inactivated (e.g., that loses a particular enzymatic or functional activity) is within the scope of the present disclosure.

The pathogens and antigens, which are described in more detail in US-2003-0175292-A1, include human immunodeficiency viruses of any clade (e.g. from any known clade or from any isolate (e.g., clade A, AG, B, C, D, E, F, G, H, I, J, K, or L). Additional HIV sequences and mutant sequences are known in the art (e.g., the HIV Sequence Database in Los Alamos and the HIV RT/Protease Sequence Database in Stanford). When the vectors include sequences from a pathogen, they can be administered to a patient to elicit an immune response. Thus, methods of administering antigen-encoding vectors, alone or in combination with one another, are also described herein. These methods can be carried out to either immunize patients (thereby reducing the patient's risk of becoming infected) or to treat patients who have already become infected; when expressed, the antigens may elicit both cell-mediated and humoral immune responses that may substantially prevent the infection (e.g., immunization can protect against subsequent challenge by the pathogen) or limit the extent of the impact of an infection on the patient's health. While in many instances the patient will be a human patient, the disclosure is not so limited. Other animals, including non-human primates, domesticated animals, and livestock can also be treated.

The compositions described herein, regardless of the pathogen or pathogenic subtype (e.g., the HIV clade(s)) they are directed against, can include a nucleic acid vector (e.g., a plasmid). As noted herein, vectors having one or more of the features or characteristics

(particularly the oriented termination sequence and a strong promoter) of the plasmids designated pGAl, pGA2 (including, of course, those vectors per se), can be used as the basis for a vaccine or therapy. Such vectors can be engineered using standard recombinant techniques (several of which are illustrated in the examples, below) to include insert sequences that encode viral antigens that, when administered to, and subsequently expressed in, a patient will elicit (e.g., induce or enhance) an immune response that provides the patient with some form of protection against the virus from which the antigens were obtained or derived (e.g., protection against infection, protection against disease, or amelioration of one or more of the signs or symptoms of a disease).

In the event the vector administered is a pGA vector, it can comprise the sequence of, for example, pGAl (SEQ ID NO: 1 of PCT/US2011/025422) or derivatives thereof (e.g., SEQ ID NOs:2 and 3 of PCT/US2011/025422), or pGA2 (SEQ ID NO:4 of PCT/US2011/025422) or derivatives thereof (e.g., SEQ ID NOs:5 and 6 of PCT/US2011/025422). The pGA vectors are described in more detail in PCT/US2011/025422 the disclosure of which is incorporated herein by reference in its entirety. pGAl is a 3897 bp plasmid that includes a promoter (bp 1-690), the CMV-intron A (bp 691-1638), a synthetic mimic of the tPA leader sequence (bp 1659 - 1721), the bovine growth hormone polyadenylation sequence (bpl761-1983), the lambda TO terminator (bp 1984-2018), the kanamycin resistance gene (bp 2037-2830) and the ColEI origin of replication (bp 2831-3890). The DNA sequence of the pGAl construct is provided in SEQ ID NO:l of PCT/US2011/025422. The indicated restriction sites are useful for cloning antigen- encoding sequences. The Cla I or BspD I sites are used when the 5 ' end of a vaccine insert is cloned upstream of the tPA leader. The Nhe I site is used for cloning a sequence in frame with the tPA leader sequence. The sites listed between Sma I and Bin I are used for cloning the 3' terminus of an antigen-encoding sequence. pGA2 is a 2947 bp plasmid lacking the 947 bp of intron A sequences found in pGAl . pGA2 is the same as pGAl , except for the deletion of intron A sequences. pGA2 is valuable for cloning sequences which do not require an upstream intron for efficient expression, or for cloning sequences in which an upstream intron might interfere with the pattern of splicing needed for good expression. A schematic map of pGA2 with useful restriction sites for cloning vaccine inserts and the DNA sequence of pGA2 is provided in SEQ ID NO:2 of

PCT/US2011/025422. The use of restriction sites for cloning vaccine inserts into pGA2 is the same as that used for cloning fragments into pGAl . pGA2.1 and pGA2.2 are multiple cloning site derivatives of pGA2. The DNA sequence of pGA2.1 is provided in SEQ ID NO:5 of PCT/US2011/025422 and the sequence of pGA2.2 is provided in SEQ ID NO:6 of

PCT/US2011/025422.

Plasmids having "backbone" sequences that differ from those disclosed herein are also within the scope of the disclosure so long as the plasmids retain substantially all of the characteristics necessary to be therapeutically effective (e.g., one can substitute nucleotides, add nucleotides, or delete nucleotides so long as the plasmid, when administered to a patient reactivates the expression of latent proviral DNA. For example, 1-10, 11-20, 21-30, 31-40, 41- 50, 51-60, 61-70, 71-80, 81-90, 91-100, or more than 100 nucleotides can be deleted or replaced.

In one embodiment, the encoded antigens can be of any HIV clade or subtype or any recombinant form thereof. With respect to inserts from immunodeficiency viruses, different clades exhibit isolate diversity, with each isolate within a clade having overall similar diversity from the consensus sequence for the clade (see, e.g., Subbarao et al, AIDS 10(Suppl A) :S 13-23, 1996). Thus, most isolates can be used as a reasonable representative of sequences for other isolates of the same clade.

Accordingly, the nucleic acid shock agents described herein can be practiced with natural variants of genes or nucleic acid molecules that result from recombination events, alternative splicing, or mutations (these variants may be referred to herein simply as "recombinant forms" of HIV).

Moreover, one or more of the viral nucleic acid inserts within any construct can be mutated to decrease their natural biological activity (and thereby increase their safety) in humans. In one embodiment, the shock agent is an HIV DNA vaccine including:

a prokaryotic origin of replication;

a selectable marker gene; and

a eukaryotic transcription cassette comprising a vaccine insert encoding one or more immunogens selected from the group consisting of HIV Gag, Pol, Tat, Rev, Vpu and Env, wherein the insert sequence encoding HIV Gag comprises one or more mutations that disable the zinc fingers and wherein the reverse transcriptase activity of HIV pol is inactivated and the protease activity is inactivated.

Other vectors of the disclosure include plasmids encoding a Gag protein (e.g., a Gag protein in which one or both of the zinc fingers have been inactivated); a Pol protein (e.g., a Pol protein in which integrase, RT, and/or protease activities have been inhibited); a Vpu protein (which may be encoded by a sequence having a mutant start codon); and Env, Tat, and/or Rev proteins (in a wild type or mutant form). As is true for plasmids encoding other antigens, plasmids encoding the antigens just described can be combined with (e.g., mixed with) other plasmids that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof). The inserts per se (sans vector) are also within the scope of the disclosure.

Particular inserts and insert-bearing compositions include the following. Where the composition includes either a vector with an insert or an insert alone, and that insert encodes a single antigen, the antigen can be a wild type or mutant gag sequence (e.g., a gag sequence having a mutation in one or more of the sequences encoding a zinc finger at one or more of the cysteine residues corresponding to clade B HIV positions 392, 395, 413, or 416 to another residue (e.g., serine) or the mutation can change one or more of the cysteine residues

corresponding to clade A/G or C positions 390, 393, 411, or 414 to another residue (e.g., serine) wherein these mutations inactivate both zine fingers. In another embodiment, the one or more immunogens includes HIV Gag and wherein the

HIV gag mutations consist of amino acid mutations corresponding to C392S, C395S, C413S, and C416S of Clade B HIV strain HXB2 that disable the zinc fingers.

The compositions used in the methods described can also include a vector (e.g., a plasmid vector) encoding: (a) a Gag protein in which one or both zinc fingers have been inactivated; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence, (ii) the polymerase, strand transfer, and/or R ase H activity of reverse

transcriptase has been inhibited by one or more point mutations within the pol sequence and (iii) the proteolytic activity of the protease has been inhibited by one or more point mutations; and (c) Env, Tat, Rev, and Vpu, with or without mutations. As noted above, proteolytic activity can be inhibited by introducing a mutation at positions 1641 - 1643 of SEQ ID NO : 8 of WO2011103417 or at an analogous position in the sequence of another HIV clade.

In one embodiment, the reverse transcriptase of Pol comprises amino acid mutations corresponding to D185N, W266T and E478Q of HIV strain HXB2 that inactivate reverse transcriptase activity.

In another embodiment, the one or more immunogens includes HIV Gag and wherein the active site of the protease was mutated to limit proteolytic cleavage of viral Gag proteins and the maturation of viral particles.

In another embodiment, the protease mutation corresponds to D25A of Clade B HIV strain HXB2.

In another embodiment, the HIV DNA vaccine includes ADA Env, Tat, Rev and Vpu, wherein the Bglll fragment in the ADA Env is deleted leaving tat, rev and vpu coding regions intact.

In another embodiment, the HIV DNA vaccine includes one or more immunogens that are clade B HIV proteins.

In another embodiment, the HIV DNA vaccine includes clade B HIV proteins, HIV

HXB2, HIV BH10 or HIV ADA proteins. In another embodiment, the vector lacks LTRs, integrase, vif, and vpr sequences of HIV.

In another embodiment, the HIV DNA vaccine includes cytomegalovirus immediate early promoter (CMVIE) and a polyadenylation sequence.

In another embodiment, the HIV DNA vaccine includes intron A of the CMV promoter. In another embodiment, the HIV DNA vaccine includes a leader sequence.

In another embodiment, the leader sequence is a polyadenylation sequence which is a bovine growth hormone polyadenylation sequence or a rabbit beta globin polyadenylation sequence.

In another embodiment, expression of the HIV DNA vaccine forms immature virus- like particles (VLPs) that bud from the plasma membrane of DNA-expressing cells.

One such exemplary HIV DNA vaccine insert encoding these above mentioned clade B zinc finger, reverse transcriptase and protease mutations and is designated herein as a "JS7 insert". An HIV DNA vaccine containing this JS7 insert in plasmid pGA2 is referred to as "GEO-D02".

At least one of the two or more sequences can be mutant or mutated so as to limit the encapsidation of viral RNA (preferably, the mutation(s) limit encapsidation appreciably). One can introduce mutations and determine their effect (on, for example, expression or

immunogenicity) using techniques known in the art; antigens that remain well expressed (e.g., antigens that are expressed about as well as or better than their wild type counterparts), but are less biologically active than their wild type counterparts, are within the scope of the disclosure. Techniques are also available for assessing the immune response. One can, for example, detect anti-viral antibodies or virus-specific T cells. Desirably, the mutant vectors or vaccine inserts provided result in an increase (e.g., at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, or 50%) in the avidity of immunogen-specific antibodies, an increase (e.g., by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, 300- fold, 400-fold, or 500-fold) in immunogen-specific antibody titers, an increase (e.g., at least 1- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, or 15-fold) in immunogen-specific IgA levels (e.g., IgA levels in rectal secretions), a level of between 0.03 to 0.3 ng of immunogen-specific IgA per μg of total IgA, a level of between 0.1 to 0.3 ng of immunogen-specific IgA per μg of total IgA, a level of between 0.2 to 0.3 ng of immunogen-specific IgA per μg of total IgA, an increase (e.g., at least 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) in resistance to HIV infection, an increase (e.g., by at least 10%>, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 1 10%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%,

270%), 280%), 290%), or 300%)) in antibody-dependent cellular cytotoxicity, an increase (e.g., at least 5%, 10%>,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%), 90%), 95%), or 100%)) in immunogen-specific CD4 helper T cells, and/or an increase (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%o, 90%), 95%), or 100%) in immunogen-specific CD8 cytotoxic T cells, and/or an increase (e.g., by at least 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21- fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 40-fold, 50- fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold) in neutralizing antibody titers. In one embodiment, the mutant constructs (e.g., a vaccine insert) can include sequences encoding one or more of the substitution mutants described herein (see, e.g. the Examples) or an analogous mutation in another HIV clade. In addition to, or alternatively, HIV antigens can be rendered less active by deleting part of the gene sequences that encode them. Thus, the compositions of the disclosure can include constructs that encode antigens that, while capable of eliciting an immune response, are mutant (whether encoding a protein of a different length or content than a corresponding wild type sequence) and thereby less able to carry out their normal biological function when expressed in a patient. As noted above, expression, immunogenicity, and activity can be assessed using standard techniques in molecular biology and immunology.

As described further below, the vectors of the present disclosure can be administered with an adjuvant, including a genetic adjuvant. Accordingly, the nucleic acid vectors, regardless of the antigen they express, can optionally include such genetic adjuvants as GM-CSF, IL-15, IL-2, interferon response factors, secreted forms of flt-3, CD40 ligand and mutated caspase genes. Genetic adjuvants can also be supplied in the form of fusion proteins, for example by fusing one or more C3d gene sequences (e.g., 1-3 (or more) C3d gene sequences) to an expressed antigen. In one embodiment, the DNA vectors express HIV-1 antigens and GM-CSF, and those constructs can be administered to subjects as described herein. The GM-CSF sequence can be introduced into a variety of different DNA vectors expressing HIV-1 antigens. HIV inserts such as those described herein, and in US-2003-0175292-A1 are particularly useful. Any plasmid within the scope of the disclosure can be tested for expression by transfecting cells, such as 293T cells (a human embryonic kidney cell line) and assessing the level of antigen expression (by, for example, an antigen-capture ELISA or a Western blot).

The GM-CSF sequence included in the vectors and the vaccine inserts may be a full- length human GM-CSF or may be a polypeptide that includes a sequence that is at least 95% identical to GM-CSF and has one or more (e.g., two or three) biological activities of GM-CSF (e.g., capable of stimulating macrophage differentiation and proliferation, or activating antigen presenting dendritic cells). The GM-CSF may include one or more mutations (e.g., one or more (e.g., at least two, three, four, five, or six) amino acid substitutions, deletions, or additions)). Desirably, any mutant GM-CSF proteins also have one or more (e.g., two or three) biological activities of GM-CSF (as described above). Assays for the measurement of the biological activity of GM-CSF proteins are known in the art (see, e.g., U.S. Patent No. 7,371,370; incorporated herein by reference in its entirety).

The nucleic acid vectors of the disclosure encode GM-CSF and at least one antigen (which may also be referred to as an immunogen) obtained from, or derived from, any HIV clade or isolate (i.e., any subtype or recombinant form of HIV). The antigen (or immunogen) may be: a structural component of an HIV virus; glycosylated, myristoylated, phosphorylated, or otherwise post-translationally modified; one that is expressed intracellularly, on the cell surface, or secreted (antigens that are not normally secreted may be linked to a signal sequence that directs secretion). More specifically, the antigen can be all, or an antigenic portion of, Gag, , Pol, Env (e.g., gpl60 or gpl20, or a CCR5-using Env), Tat, Rev, Vpu, Nef, Vif, Vpr, or a VLP (e.g., a polypeptide derived from a VLP that is capable of forming a VLP, including an Env-defective HIV VLP).

In one embodiment, the HIV DNA vaccine contains the above-described JS7 insert and also a sequence encoding GM-CSF as described in WO2011103417. This HIV DNA vaccine is also designated "GEO-D03". Multiclade Nucleic Acid Vaccines

Where the composition includes either a vector with an insert or an insert alone, and that insert encodes multiple protein antigens, one of the antigens can be a wild type or mutant gag sequence, including those described above. Similarly, where a composition includes more than one type of vector or more than one type of insert, at least one of the vectors or inserts (whether encoding a single antigen or multiple antigens) can include a wild type or mutant gag sequence, including those described above or analogous sequences from other HIV clades. For example, where the composition includes first and second vectors, the vaccine insert in either or both vectors (whether the insert encodes single or multiple antigens) can encode gag; where both vectors encode gag, the gag sequence in the first vector can be from one HIV clade (e.g., clade B) and that in the second vector can be from another HIV clade (e.g., clade C).

Where the composition includes either a vector with an insert or an insert alone, and that insert encodes a single antigen, the antigen can be wild type or mutant Pol. The sequence can be mutated by deleting or replacing one or more nucleic acids, and those deletions or substitutions can result in a Pol gene product that has less enzymatic activity than its wild type counterpart

(e.g., less integrase activity, less reverse transcriptase (RT) activity, or less protease activity). For example, one can inhibit RT by inactivating the polymerase's active site or by ablating strand transfer activity. Alternatively, or in addition, one can inhibit the polymerase's RNase H activity. Where the composition includes either a vector with an insert or an insert alone, and that insert encodes multiple protein antigens, one of the antigens can be a wild type or mutant pol sequence, including those described above (these multi-protein-encoding inserts can also encode the wild type or mutant gag sequences described above). Similarly, where a composition includes more than one type of vector or more than one type of insert, at least one of the vectors or inserts (whether encoding a single antigen or multiple antigens) can include a wild type or mutant pol sequence, including those described above (and, optionally, a wild type or mutant gag sequence, including those described above (i.e., the inserts can encode Gag-Pol). For example, where the composition includes first and second vectors, the vaccine insert in either or both vectors (whether the insert encodes single or multiple antigens) can encode Pol; where both vectors encode Pol, the Pol sequence in the first vector can be from one HIV clade (e.g., clade B) and that in the second vector can be from another HIV clade (e.g., clade A or G). Where an insert includes some or all of the pol sequence, another portion of the pol sequence that can optionally be altered is the sequence encoding the protease activity (regardless of whether or not sequences affecting other enzymatic activities of Pol have been altered). Where the composition includes either a vector with an insert or an insert alone, and that insert encodes a single antigen, the antigen can be a wild type or mutant Env, Tat, Rev, Nef, Vif, Vpr, or Vpu. Where the composition includes either a vector with an insert or an insert alone, and that insert encodes multiple protein antigens, one of the antigens can be a wild type or mutant Env. For example, multi-protein expressing inserts can encode wild type or mutant Gag-Pol and Env; they can also encode wild type or mutant Gag-Pol and Env and one or more of Tat, Rev, Nef, Vif, Vpr, or Vpu (each of which can be wild type or mutant). As with other antigens, Env, Tat, Rev, Nef, Vif, Vpr, or Vpu can be mutant by virtue of a deletion, addition, or substitution of one or more amino acid residues (e.g., any of these antigens can include a point mutation). For example, one or more amino acids can be deleted from the gpl20 surface and/or gp41 transmembrane cleavage products of Env. With respect to Gag, one or more amino acids can be deleted from one or more of: the matrix protein (p7), the capsid protein (p24), the nucleocapsid protein (p7) and the C-terminal peptide (p6). For example, amino acids in one or more of these regions can be deleted (this may be especially desired where the vector is a viral vector, such as MVA). With respect to Pol, one or more amino acids can be deleted from the protease protein (plO), the reverse transcriptase protein (p66/p51), or the integrase protein (p32). More specifically, the compositions of the disclosure can include a vector (e.g., a plasmid or viral vector) that encodes: (a) a Gag protein in which one or more of the zinc fingers has been inactivated to limit the packaging of viral R A; (b) a Pol protein in which (i) the integrase activity has been inhibited by deletion of some or all of the pol sequence and (ii) the polymerase, strand transfer, and/or RNase H activity of reverse transcriptase has been inhibited by one or more point mutations within the pol sequence; and (c) Env, Tat, Rev, and Vpu, with or without mutations. In this embodiment, as in others, the encoded proteins can be obtained or derived from a subtype A, B or C HIV (e.g., HIV-1) or recombinant forms thereof. Where the compositions include non- identical vectors, the sequence in each type of vector can be from a different HIV clade (or subtype or recombinant form thereof). For example, the disclosure features compositions that include plasmid vectors encoding the antigens just described (Gag-Pol, Env etc.), where some of the plasmids include antigens that are obtained from, or derived from, one clade and other plasmids include antigens that are obtained (or derived) from another clade. Mixtures representing two, three, four, five, six, or more clades (including all clades) are within the scope of the disclosure.

Where first and second vectors are included in a composition, either vector can be pGAl/JS2, pGAl/JS7, pGAl/JS7.1 , pGA2/JS2, pGA2/JS7, pGA2/JS7.1 (pGAl .1, pGAl .2 or the pGA vectors with other permutations in restrictions sites used for addition of vaccine inserts can be used in place of pGAl, and pGA2.1 or pGA2.2 can be used in place of pGA2). Similarly, either vector can be pGAl/IC25, pGAl/IC2, pGAl/IC48, pGAl/IC90, pGA2/IC25, pGA2/IC2, pGA2/IC48, or pGA2/IC90 (here again, pGAl . 1 or pGA1.2 can be used in place of pGAl, and pGA2.1 or pGA2.2 can be used in place of pGA2). In alternative embodiments, the encoded proteins can be those of, or those derived from, a subtype C HIV (e.g.,, HIV1) or a recombinant form thereof. For example, the vector can be pGAl/IN2, pGAl . 1/IN2, pGA1.2/IN2, pGAl/IN3, pGAl .1/IN3, pGA1.2/IN3, pGA2/IN2, pGA2.1/IN2, pGA2.2/IN2, pGA2/IN3, pGA2.1/IN3, or pGA2.2/IN3. These vectors are described in WO2003076591 and WO2011103417 incorporated herein by reference in their entirety.

The encoded proteins can also be those of, or those derived from, any of HIV clades (or subtypes) E, F, G, H, I, J, K or L or recombinant forms thereof. An HIV-1 classification system has been published by Los Alamos National Laboratory (HIV Sequence Compendium-2001 , Kuiken ct al, published by Theoretical Biology and Biophysics Group T-10, Los Alamos, NM, (2001 )), more recent HIV sequences are available on the Los Alamos HIV sequence database website.

For example, the plasmids can contain the inserts such as JS7, IC25, and IN3 referenced in WO2003076591 and shown in Table 1.

Table 1

IC90 Gag C390S AG HIV-1 Isolate 928

Gag C393S

Gag C411S

Gag C414S

Protease L90M

RT D185N

RT W266T

RNaseH E478Q

IN3 Gag C390S Clade C HIV-1 Isolate

Gag C393S 98IN012

Gag C411S

Gag C414S

Protease D25A

RT D185N

RT W266T

RNaseH E478Q

IN2 Gag C390S Clade C HIV-1 Isolate

Gag C393S 98IN012

Gag C411S

Gag C414S

RT D185N

RT W266T

RNaseH E478Q

As is true for plasmids encoding other antigens, plasmids encoding the antigens just described can be combined with (e.g., mixed with) other plasmids that encode antigens obtained from, or derived from, a different HIV clade (or subtype or recombinant form thereof). The inserts per se (sans vector) are also within the scope of the disclosure. As described herein, the inserts may contain sequences that encode one or more conserved protein sequences and/or may contain one or more designer sequences (e.g., mosaic sequences that contain a sequence from one or more HIV clades).

The plasmids described above, including those that express the JS2 or JS7 series of clade B HIV-1 sequences, can be administered to any subject as shock agents. Similarly, plasmids or other vectors that express an IN series of clade C HIV-1 sequences can be administered as a shock agent to a subject who has been infected with an HIV of clade C or another clade. As vectors expressing antigens of various clades can be combined to elicit an immune response against more than one clade (this can be achieved whether one vector expresses multiple antigens, or mosaic or conserved element antigens from different clades or multiple vectors express single antigens from different clades), one can tailor the vaccine formulation to best protect a given subject. The antigens they express are not the only parts of the plasmid vectors that can vary. Useful plasmids may or may not contain a terminator sequence that substantially inhibits transcription (the process by which R A molecules are formed upon DNA templates by complementary base pairing). Useful terminator sequences include the lambda TO terminator and functional fragments or variants thereof. The terminator sequence is positioned within the vector in the same orientation and at the C terminus of any open reading frame that is expressed in prokaryotes (i.e., the terminator sequence and the open reading frame are operably linked). By preventing read through from the selectable marker into the vaccine insert as the plasmid replicates in prokaryotic cells, the terminator stabilizes the insert as the bacteria grow and the plasmid replicates.

Selectable marker genes are known in the art and include, for example, genes encoding proteins that confer antibiotic resistance on a cell in which the marker is expressed (e.g., resistance to kanamycin, ampicillin, or penicillin). The selectable marker is so-named because it allows one to select cells by virtue of their survival under conditions that, absent the marker, would destroy them. The selectable marker, the terminator sequence, or both (or parts of each or both) can be, but need not be, excised from the plasmid before it is administered to a patient. Similarly, plasmid vectors can be administered in a circular form, after being linearized by digestion with a restriction endonuclease, or after some of the vector "backbone" has been altered or deleted.

The nucleic acid vectors can also include an origin of replication (e.g., a prokaryotic origin of replication) and a transcription cassette that, in addition to containing one or more restriction endonuclease sites, into which an antigen-encoding insert can be cloned, optionally includes a promoter sequence and a polyadenylation signal. Promoters known as strong promoters can be used and may be preferred. One such promoter is the cytomegalovirus (CMV) intermediate early promoter, although other (including weaker) promoters may be used without departing from the scope of the present disclosure. Similarly, strong polyadenylation signals may be selected (e.g., the signal derived from a bovine growth hormone (BGH) encoding gene, or a rabbit β globin polyadenylation signal (Bohm et al, J. Immunol. Methods 193:29-40, 1996; Chapman et al, Nucl. Acids Res. 19:3979- 3986, 1991; Hartikka et al, Hum. Gene Therapy 7: 1205-1217, 1996; Manthorpe et al, Hum. Gene Therapy 4:419-431, 1993; Montgomery et al, DNA Cell Biol. 12:777-783, 1993)).

The vectors can further include a leader sequence (a leader sequence that is a synthetic homolog of the tissue plasminogen activator gene leader sequence (tPA) is optional in the transcription cassette) and/or an intron sequence, such as a cytomegalovirus (CMV) intron A or an SV40 intron. The presence of intron A increases the expression of many antigens from RNA viruses, bacteria, and parasites, presumably by providing the expressed RNA with sequences that support processing and function as a eukaryotic mRNA.

Expression can also be enhanced by other methods known in the art including, but not limited to, optimizing the codon usage of prokaryotic mRNAs for eukaryotic cells (Andre et al, J. Virol. 72: 1497-1503, 1998; Uchijima et al, J. Immunol. 161 :5594-5599, 1998). Multi- cistronic vectors may be used to express more than one immunogen or an immunogen and an immunostimulatory protein (Iwasaki et al., J. Immunol. 158:4591-4601, 1997a; Wild et al, Vaccine 16:353-360, 1998). Thus (and as is true with other optional components of the vector constructs), vectors encoding one or more antigens from one or more HIV clades or isolates may, but do not necessarily, include a leader sequence and an intron (e.g., the CMV intron A). The vectors of the present disclosure differ in the sites that can be used for accepting antigen-encoding sequences and in whether the transcription cassette includes intron A sequences in the CMVIE promoter. Accordingly, one of ordinary skill in the art may modify the insertion site(s) or cloning site(s) within the plasmid without departing from the scope of the disclosure. Both intron A and the tPA leader sequence have been shown in certain instances to enhance antigen expression (Chapman et al, Nucleic Acids Research 19:3979-3986, 1991).

In one embodiment, the shock agent can include any viral or bacterial vector that includes an insert described herein. The disclosure, therefore, also encompasses administration of at least two (e.g., three, four, five, or six) vectors (e.g., plasmid or viral vectors that contain the same vaccine insert (i.e., an insert encoding the same antigens). As is made clear elsewhere, the patient may receive two types of vectors, and each of those vectors can elicit an immune response against an HIV of a different clade. For example, the disclosure features methods in which a patient receives a composition that includes (a) a first vector comprising a vaccine insert encoding one or more antigens that elicit an immune response against a human

immunodeficiency virus (HIV) of a first subtype or recombinant form and (b) a second vector comprising a vaccine insert encoding one or more antigens that elicit an immune response against an HIV of a second subtype or recombinant form. The first and second vectors can be any of those described herein. Similarly, the inserts in the first and second vectors can be any of those described herein.

At least some of the immunodeficiency virus vaccine inserts of the present disclosure were designed to generate non-infectious VLPs (a term that can encompass true VLPs as well as aggregates of viral proteins) from a single DNA. This was achieved using the subgenomic splicing elements normally used by immunodeficiency viruses to express multiple gene products from a single viral R A. The subgenomic splicing patterns are influenced by (i) splice sites and acceptors present in full length viral RNA, (ii) the Rev responsive element (RRE) and (iii) the Rev protein. The splice sites in retroviral RNAs use the canonical sequences for splice sites in eukaryotic mRNAs. The RRE is an approximately 200 bp RNA structure that interacts with the Rev protein to allow transport of viral RNAs from the nucleus to the cytoplasm. In the absence of Rev, the approximately 10 kb RNA of immunodeficiency virus mostly undergoes splicing to the mRNAs for the regulatory genes Tat, Rev, and Nef. These genes are encoded by exons present between RT and Env and at the 3' end of the genome. In the presence of Rev, the singly spliced mRNA for Env and the unspliced mRNA for Gag and Pol are expressed in addition to the multiply spliced mRNAs for Tat, Rev, and Nef.

The expression of non-infectious VLPs from a single DNA affords a number of advantages to an immunodeficiency virus vaccine. The expression of a number of proteins from a single DNA affords the vaccinated host the opportunity to respond to the breadth of T- and B- cell epitopes encompassed in these proteins. The expression of proteins containing multiple epitopes allows epitope presentation by diverse histocompatibility types. By using whole proteins, one offers hosts of different histocompatibility types the opportunity to raise broad- based T cell responses. This may be essential for the effective containment of immunodeficiency virus infections, whose high mutation rate supports ready escape from immune responses (Evans et al, Nat. Med. 5: 1270-1276, 1999; Poignard et al, Immunity 10:431-438, 1999, Evans et al, 1995). In the context of the present shock/kill treatment, just as in drug therapy, multi-epitope T cell responses that require multiple mutations for escape will provide better protection than single epitope T cell responses (which require only a single mutation for escape).

Immunogens can also be engineered to be more or less effective for raising antibody or T cells by targeting the expressed antigen to specific cellular compartments. For example, antibody responses are raised more effectively by antigens that are displayed on the plasma membrane of cells, or secreted therefrom, than by antigens that are localized to the interior of cells (Boyle et al, Int. Immunol. 9: 1897-1906, 1997; Inchauspe et al, DNA Cell. Biol. 16: 185-195, 1997). Tc responses may be enhanced by using N-terminal ubiquitination signals which target the DNA encoded protein to the proteosome causing rapid cytoplasmic degradation and more efficient peptide loading into the MHC I pathway (Rodriguez et al, J. Virol. 71 :8497-8503, 1997; Tobery et al, J. Exp. Med. 185:909-920, 1997; Wu et al, J. Immunol. 159:6037-6043, 1997). For a review on the mechanistic basis for DNA-raised immune responses, refer to Robinson and Pertmer, Advances in Virus Research, vol. 53, Academic Press (2000). CpG oligodeoxynucleotides

In another embodiment, the shock agent is a CpG oligodeoxynucleotide (CpG ODN), also referred to as a CpG oligonucleotide. Toll-like receptor 9 (TLR9) senses unmethylated CpG dinucleotides, a hallmark of microbial DNA, that can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs). In one embodiment, the CpG oligonucleotide is co- administered with an HIV DNA vaccine as the shock agent.

TLR9 stimulation by CpG DNA or CpG ODNs triggers intracellular signaling leading to the activation of macrophages, dendritic cells (DC) and B cells, and the production of cytokines, chemokines, and immunoglobulins. Subsequently, cytokines produced by DC, such as IL-12, induce the differentiation of naive T cells into T helper 1 (Thl) and cytotoxic T-cells (CTL). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated

CpG dinucleotides in particular sequence contexts (CpG motifs). For stability, CpG ODNs may possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone found in genomic bacterial DNA. Three major classes of stimulatory CpG ODNs have been identified based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These three classes are Class A (Type D), Class B (Type K) and Class C. CpG-A ODNs are characterized by a PO central CpG-containing palindromic motif and a

PS-modified 3' poly-G string. They induce high IFN-a production from pDCs but are weak stimulators of TLR9-dependent NF-κΒ signaling and pro-inflammatory cytokine (e.g. IL-6) production.

CpG-B ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κΒ signaling but weakly stimulate IFN-a secretion.

CpG-C ODNs combine features of both classes A and B. They contain a complete PS backbone and a CpG-containing palindromic motif. C-Class CpG ODNs induce strong IFN-a production from pDC as well as B cell stimulation. In one particular embodiment, the CpG oligonucleotide is CpG 7909. CpG 7909 is an immunomodulating synthetic oligonucleotide designed to specifically activate the Toll-like receptor 9 (TLR9). CpG 7909, acting through the TLR9 receptor present in B cells and plasmacytoid dendritic cells, stimulates human B-cell proliferation, enhances antigen-specific antibody production and induces interferon-alpha production, interleukin-10 secretion and natural killer cell activity.

In another embodiment, the CpG ODN is CpG 2006. CpG 2006 is a Class B synthetic oligonucleotide, also known to specifically activate the Toll-like receptor 9 (TLR9).

In other embodiments, the CpG ODN is another synthetic oligonucleotide, for example CpG ODN 1585, CpG ODN 2118, CpG ODN 1826, or CpG 1668. In another embodiment, the shock agent is an HDAC inhibitor.

In one embodiment the HDAC inhibitor is a Class I HDAC inhibitor. In another embodiment, the Class I HDAC inhibitor is selected from HDAC1, HDAC 2, HDAC 3 and HDAC 8. In one embodiment the HDAC inhibitor is a Class II HDAC inhibitor. In another embodiment, the Class II HDAC inhibitor is selected from HDAC4, HDAC 5, HDAC 6, HDAC 7, HDAC9 and HDAC 10.

In one embodiment the HDAC inhibitor is a Class III HDAC inhibitor, also known as the sirtuins. In another embodiment, the Class III HDAC inhibitor is selected from SIRTl, SIRT 2, SIRT 3, SIRT 4, SIRT 5, SIRT 6, and SIRT 7.

In one embodiment the HDAC inhibitor is a Class IV HDAC inhibitor., In another embodiment, the Class IV HDAC inhibitor is HDAC11.

In one embodiment the HDAC inhibitor is selected from Romidepsin (Istodax, depsipeptide, NSC 630176, FK228 or FR901228), Panobinostat (LBH589), Valproic acid,

Belinostat (PXDIOI), Mocetinostat (MGCD0103), Abexinostat (PCI-24781), Entinostat (MS- 275), SB939, Resminostat (4SC-201) , Givinostat (ITF2357), Quisinostat (JNJ-26481585), CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, Kevetrin, Trichostatin A (TSA). In another embodiment, the shock agent is an NFkappaB activator such as, but not limited to, phorbol esters and TNF-a.

In another embodiment, the shock agent is a cytosine methylation inhibitor such as, but not limited to 5-azacytidine.

In another embodiment, the shock agent is an acetaldehyde dehydrogenase inhibitor. In one embodiment, the acetaldehyde dehydrogenase inhibitor is selected from 4-amino-

4-methyl-2-pentyne-l-al, Benomyl, Chloral, Chlorpropamide analogs, Citral, Coprine,

Cyanamide, Daidzin (antioxidant isoflavone), 4-(Diethylamino)benzaldehyde, Disulfiram, Gossypol, Kynurenine metabolites, Molinate, Nitroglycerin, or Pargyline.

In a particular embodiment, the acetaldehyde dehydrogenase inhibitor is disulfiram. In one embodiment, combinations of the above described shock agents may be used to optimize the reliability of reservoir activation. In one embodiment, the HIV DNA vaccine may be combined with one or more of a CpG oligonucleotide, a TLR9 receptor agonist, an HDAC inhibitor, an NFkappaB activator, a cytosine methylation inhibitor, or an acetaldehyde dehydrogenase inhibitor.

In one particular embodiment, a CpG oligodeoxynucleotide can be used in combination with the HIV DNA vaccine to shock viral reservoirs.

II. Kill Agents

As used herein the term "kill agent" refers to a composition useful in killing, destroying, eradicating, or lysing virus-expressing cells. In a particular embodiment, kill agents target and destroy reactivated CD4+ HIV reservoirs. The kill agent may be any suitable agent, such as a viral vector, viral vaccine or antibody, e.g., monoclonal antibody or recombinant antibody.

A. HIV Vaccines

In some embodiments, the HIV vaccine is a HIV vaccine that elicits a T cell immune response. Methods of measuring T cell immune response are known in the art. In specific embodiments, the HIV vaccine is selected from an MVA vaccine, an adenoviral vaccine, or a vesicular stomatitis virus (VSV) vaccine. Such vaccines are known to those of skill in the art. (See Schiff er et al, Retrovirology, 2013, 10:72)

In one particular embodiment, the HIV vaccine that may be used as the kill agent of the present methods is a modified vaccinia Ankara (MVA) vaccine. Such MVA HIV vaccines are known in the art and have been described in international patent applications WO2002/072754 and WO/2006/026667.

MVA has been particularly effective as a vaccine in mouse models (Schneider et al., Nat. Med. 4:397-402, 1998). MVA is a highly attenuated strain of vaccinia virus that was developed toward the end of the campaign for the eradication of smallpox, and it has been safety tested in more than 100,000 people (Mahnel et al, Berl. Munch Tierarztl Wochenschr 107:253-256, 1994; Mayr et al, Zentralbl. Bakteriol. 167:375-390, 1978). During over 500 passages in chicken cells, MVA lost about 10% of its genome and the ability to replicate efficiently in primate cells.

Despite its limited replication, MVA has proved to be a highly effective expression vector (Sutter et al, Proc. Natl. Acad. Sci. U.S.A. 89: 10847-10851,1992), raising protective immune responses in primates for parainfluenza virus (Durbin et al. J. Infect. Dis. 179: 1345-1351, 1999), measles (Stittelaar et al. J. Virol. 74:4236-4243, 2000), and immunodeficiency viruses (Barouch et al, J. Virol. 75:5151-5158, 2001; Ourmanov et al, J. Virol. 74:2740-2751, 2000; Amara et al, J. Virol. 76:7625-7631, 2002). The relatively high immunogenicity of MVA has been attributed in part to the loss of several viral anti-immune defense genes (Blanchard et al, J. Gen. Virol. 79: 1159-1167, 1998).

Vaccinia viruses have been used to engineer viral vectors for recombinant gene expression and as recombinant live vaccines (Mackett et al, Proc. Natl. Acad. Sci. U.S.A.

79:7415-7419; Smith et al, Biotech. Genet. Engin. Rev. 2:383-407, 1984). DNA sequences, which may encode any of the HIV antigens described herein, can be introduced into the genomes of vaccinia viruses. If the gene is integrated at a site in the vector DNA that is nonessential for the life cycle of the virus, it is possible for the newly produced recombinant vaccinia virus to be infectious (i.e., able to infect cells) and to express the integrated DNA sequences. Preferably, the viral vectors featured in the compositions and methods of the present disclosure are highly attenuated. Several attenuated strains of vaccinia virus were developed to avoid undesired side effects of smallpox vaccination. The modified vaccinia Ankara (MVA) virus was generated by long-term serial passages of the Ankara strain of vaccinia virus on chicken embryo fibroblasts (CVA; see Mayr et al, Infection 3:6-14, 1975). The MVA virus is publicly available from the American Type Culture Collection (ATCC; No. VR-1508; Manassas, VA). The desirable properties of the MVA strain have been demonstrated in clinical trials (Mayr et al, Zentralbl. Bakteriol. 167:375-390, 1978; Stickl et al, Dtsch. Med. Wschr. 99:2386-2392, 1974; see also, Sutter and Moss, Proc. Natl. Acad. Sci. U.S.A. 89: 10847-10851, 1992). During these studies in over 120,000 humans, including high-risk patients, no side effects were associated with the use of MVA vaccine. The MVA vectors can be prepared as follows. A DNA construct that contains a DNA sequence that encodes a foreign polypeptide (e.g., any of the HIV antigens described herein) and that is flanked by MVA DNA sequences adjacent to a naturally occurring deletion within the MVA genome (e.g., deletion III or other non-essential site(s); six major deletions of genomic DNA (designated deletions I, II, III, IV, V, and VI) totaling 31,000 base pairs have been identified (Meyer et al, J. Gen. Virol. 72: 1031-1038, 1991)) is introduced into cells infected with

MVA under conditions that permit homologous recombination to occur. Insertions may also be introduced into naturally-occurred deletions with modified deletion sites (e.g., restructured deletion III, but not only) to enhance stability of the insertion or introduced between essential genes using sequences flanking the insertion site. One site between essential genes that has proven useful is I18R/G1LI18G1 (see, for e.g., Wyatt et al,

Retrovirology 6:416, 2009). Once the DNA construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, the recombinant vaccinia virus can be isolated by methods known in the art (isolation can be facilitated by use of a detection marker). The DNA to be inserted can be linear or circular (e.g., a plasmid, linearized plasmid, gene, gene fragment, or modified HIV genome). The foreign DNA sequence is inserted between the sequences flanking the naturally-occurring deletion, between the sequences of a modified naturally occurring deletion, or between the sequences marking the boundaries of two essential genes. For better expression of a DNA sequence, the sequence can include regulatory sequences (e.g., a promoter, such as the promoter of the vaccinia 11 kDa gene or the 7.5 kDa gene or modified H5). The DNA construct can be introduced into MVA-infected cells by a variety of methods, including calcium phosphate-assisted transfection (Graham et al, Virol. 52:456-467,

1973 and Wigler et al, Cell 16:777-785, 1979), electroporation (Neumann et al, EMBO J. 1 :841- 845, 1982), microinjection (Graessmann et al., Meth. Enzymol. 101 :482-492, 1983), by means of liposomes (Straubinger et al, Meth. Enzymol. 101 :512-527, 1983), by means of spheroplasts (Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980), or by other methods known in the art. Suitable inserts include Gag-Pol and Env sequences from the JS7, IC25 and IN3 inserts described above in Table 1.

B. Antibodies

Among potential kill agents are antibodies, including for example, neutralizing or non- neutralizing antibodies. In a particular embodiment, the kill agent is a monoclonal antibody or recombinant antibody.

In another embodiment, the kill agent is a human monoclonal or recombinant antibody.

Non-neutralizing antibodies kill by mobilizing innate immune responses such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and Complement-mediated (C '-mediated) lysis. While not to be bound by any specific mechanism, it is believed that ADCC-mediating antibodies recognize HIV-1 antigens expressed on the membrane of infected cells and bind the Fey receptor (FcR) of the effector cell population to induce ADCC.

Neutralizing Ab can elicit innate immune responses to kill viral reservoirs. In one embodiment, the kill agent is a neutralizing Ab such as 10-1074, 10E8, 17B, 2F5,

2G12, 447-52D, 448-D, 4E10, 670D, bl2, BB34, CAP206-CH12, CHOI, CH02, CH03, CH04, CH08, CH30, CH31, CH32, CH33, CH34, HJ16, M66.6, NIH45, NIH46, PG16, PG9, PGT121, PGT128, VRCOl, VRC02, VRC03 or VRC07.

In one embodiment, the kill agent is a non-neutralizing antibody such as 120-16, 126-50, 246-D, 4B3, 50-69, 98-43, 98-6, b6, 3D6, A32, or F240

In another embodiment, the killing agent is an antibody to a gpl20 carbohydrate epitope of HIV.

In a specific embodiment, this gpl20 carbohydrate epitope-binding antibody killing agent is any one of a number of broadly neutralizing MAb. In one embodiment, the Ab is A32 which is believed to bind to the CI region of gpl20.

III. Antiretro viral Therapy (ART)

The methods provided herein employ combination therapy with at least one antiretroviral agent, each of which is administered to the subject in a therapeutically effective amount. For the purposes of the present methods, ART is employed to prevent, or reduce spread of any reactivated HIV infection that could result from stimulation of viral reservoirs with shock/kill agents. The antiretroviral agent is preferably administered prior to, or during the administration of shock kill agents.

In one embodiment, the subject is undergoing a regular regimen of antiretroviral therapy prior to and during the administration of the shock and kill agents.

For purposes of the present invention, antiretroviral agents include any substance that can inhibit, reduce, or eliminate retroviral infection of a cell. As used herein ART, refers to any antiretroviral therapy including highly active antiretroviral therapy (HAART). HAART is generally considered ART therapy that comprises at least 3 antiretro viral agents. (Stebhing et al., Clin Exp Immunol. Aug 2006; 145(2): 271-276.) A number of these agents are commercially available for administration according to the manufacturer's recommended dosage. Such antiretro viral agents include, but are not limited to, the two classes known as reverse transcriptase inhibitors and protease inhibitors, as well as agents that are inhibitors of viral entry. Although any combination of these agents can be used, preferably ART comprises the administration of therapeutically effective amounts of at least one reverse transcriptase inhibitor and at least one protease mhibitor in combination with at least one additional antiretroviral agent. For example, in one embodiment of the invention, at least two reverse transcriptase inhibitors are administered in combination with at least one protease mhibitor. In another embodiment of the invention, at least two protease inhibitors are administered in combination with at least one reverse transcriptase mhibitor.

A number of reverse transcriptase inhibitors are commercially available for use in administering ART. Examples include, but are not limited to, nucleoside analogs, which are a class of compounds that are known to inhibit HIV, and non-nucleoside drugs. Nucleoside analogs are exemplified by didanosine (2',3'-dideoxyinosine or [ddl], available as Videx® from Bristol Myers-Squibb, Waliiiigford, Conn,); zidovudine (3 ^" '-azido-2',3'-dideoxythymidine or azidothymidine [AZT], available from Glaxo- Wellcome Co,, Research Triangle Park, N,C); zaicitabine (2',3 '-dideoxycytidine [ddC], available as Hivid® from Hoffman-La Roche, Basel, Switzerland); lamivudine 2'-deoxy-3'-thiacytidine [3TC] (Epivir®, available from Glaxo- Wellcome Co.); stavudine (2',3 '-didehydro-2',3 '-dideoxythimidine [D4T] available as Zerit®) from Bristol Myers-Squibb); and the combination drug zidovudine plus lamivudine (Combivir®, available from Glaxo Wellcome). These particular drugs belong to the class of compounds known as 2',3'-dideoxynucleoside analogs, which, with some exceptions such as 2',3'~ dideoxyuridine [DDU], are known to inhibit HIV replication, but have not been reported to clear any individual of the vims. Other nucleoside reverse transcriptase inhibitors include abacavir (1592U89, Ziagen™, available from Glaxo- Wellcome Co.). Non-nucleoside reverse

transcriptase inhibitors include nevirapine (Viramune™, available from Boehringer Ingeiheim Pharmaceuticals, Inc.); delaviridine (Rescriptor®, available from Pharmacia & Upjohn,

Kalamazoo, Mich.); and efavirenz (available as Sustiva®, from DuPont Merck). Examples of protease inhibitors useful in the present invention include, but are not limited to, Indinavir sulfate (available as Crixivan™ capsules from Merck & Co., Inc., West Point, Pa.), saquinavir (Invirase® and Fortovase®, available from Hoffmnan-La Roche), ritonavir (Norvir®, available from Abbott Laboratories, Abbott Park, 111.); ABT-378 (new name: lopinavir, available from Abbott Laboratories); Amprenavir (Agenerase™, available from Glaxo Wellcome, Inc.); and Nelfinavir (Viraeept®), and GWI 1 (available from Glaxo

Wellcome/Vertex).

Such examples of reverse transcriptase and protease inhibitors are not intended to be limiting. It is recognized that any known inhibitor, such as novel integrase inhibitors, as well as those under development, may be used in the methods of the invention. See, for example, the drugs for HIV infection disclosed in Medical Letter 42(Jan, 10, 2000): 1-6, herein incorporated by reference.

Suitable human dosages for these compounds can var widely. However, such dosages can readily be determined by those of skill in the art. Therapeutically effective amounts of these drugs are administered during ART. By "therapeutically effective amount" is intended an amount of the antiretroviral agent that is sufficient to decrease the effects of HIV infection, or an amount that is sufficient to favorably influence the pharmacokinetic profi le of one or more of the other antiretroviral agents used in the ART protocol. By "favorably influence" is intended that the antiretroviral agent, when administered in a therapeutically effective amount, affects the metabolism of one or more of the other antiretroviral agents used in ART, such that the bioavailability of the other agent or other agents is increased. This can allow for decreased dosage frequency of the antiretroviral agent or agents whose bioavailabi lity is increased in this manner. Decrease in dosage frequency can be advantageous for antiretroviral agents having undesirable side effects w ^r hen administered in the absence of the antiretroviral agent that increases their bioavailability. The therapeutically effective dose of an antiretroviral agent for purposes of having a favorable influence on the pharmacokinetics of another antiretroviral agent used in the ART protocol is typically lower than the amount to be administered to have a direct therapeutic effect on HIV, such as inhibition of HIV replication. When used in this manner, an antiretroviral agent that has undesirable adverse effects at the full dosage required for therapeutic effectiveness against HIV replication can provide a therapeutic benefit a lower doses with fewer adverse side effects. Thus, in one embodiment, an antiretroviral agent, when administered in a therapeutically effective amount to an HIV-infected subject, decreases the effects of HIV infection by, for example, inhibiting replication of HIV, thereby decreasing viral load in the subject undergoing antiretroviral therapy. In another embodiment, an antiretroviral agent, when administered in a therapeutically effective amount to an HIV-infected subject, favorably influences the

pharmacokinetics of one or more of the other antiretroviral agents used in the ART protocol.

For example, the protease inhibitor ritonavir when administered at full doses is a potent inhibitor of HIV in serum-and lymph nodes. When administered for these purposes, adverse reactions are common, such as gastrointestinal intolerance, hyperglycemia, insulin resistance, new onset or worsening diabetes, increased bleeding in hemophiliacs, circumoral and peripheral paresthesias, altered taste, and nausea and vomitmg. Ritonavir can be administered at low doses (for example, 100 to 400 mg bid) with minimal intrinsic antiviral activity to increase the serum concentrations and decrease the dosage frequency of other protease inhibitors (see, Hsu et al. (1998) Clin. Pharmacokinet, 35:275). See, for example, the favorable influence of ritonavir on the protease inhibitor lopinavir (ABT-378) (Eron et al. (1999) ICAAC 39 addendum: 18, Abstract LB-20).

Guidance as to dosages for any given a tiretroviral agent is available in the art and includes administering commercially available agents at their recommended dosages. See, for example, Medical Letter 42(Jan. 10, 2000): 1-6, herein incorporated by reference. Thus, for example, IDV can be administered at a dosage of about 800 mg, three times a day; D4T can be administered at a dosage of about 30-40 mg, twice a day; and Nelfinavir can be administered at a dosage of about 1250 mg, twice a day, or 750 mg three times a day. These agents are generally administered in oral formulations, though any suitable means of administration known in the art may be utilized for their delivery. For purposes of the present invention, ART ^' is administered to an HIV-infected subject to effectively reduce the pool of actively replicating virus to an undetectable amount in plasma samples collected from the subject. By "undetectable amount" in the plasma is intended the amount of actively replicating HIV is less than about 500 RNA molecules/mi, preferably less than about 400 RNA molecules/ml, more preferably less than about 300 RN A molecules/ml, still more preferably less than about 200 RNA molecules/ml, even more preferably less than about 100 RNA molecules/ml, most preferably less than about 50 RNA molecules/ml. Any method known to those skil led in the art may be utilized to measure viral load in the plasma, including the methods described elsewhere herein. Thus, for example, plasma viral load can be determined using a branched chain DNA assay (bDNA), which has a lower limit of detection (LLD) of 50 HIV RNA molecules/ml (see Jacobson et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10405-10410; herein incorporated by reference). When an undetectable amount of replicating virus is present in a plasma sample obtained from an HIV-infected subject, plasma viral RN A is said to be

"undetectable" in the subject.

IV. Methods of Use

A. Treatment Regimes

The compositions described herein are administered in a multi-step method to reduce or purge viral reservoirs for eradication by patient immune response. Stimulating and killing viral reservoirs under conditions that reduce or clear active virus out of the body provides a two-step approach for flushing or clearing viral reservoirs and preventing viral re-emergence. Slight increases in viral levels have been observed typically 1-2 weeks post DNA vaccinations in a clinical trial where combination DNA/MVA vaccines were administered in conjunction with anti-retro viral therapy (ART). Timing the DNA vaccine's effect of flushing HIV from the reservoirs as a "shock agent" and administration of a "kill agent" such as an antibody or viral vaccine in the presence of ART provides a method to reduce or eliminate viral reservoirs. While not to be bound by any particular mechanism, it is believed that a DNA vaccine acts as a "shock agent" and stimulates the reservoirs to express virus thereby making the infected cells susceptible to killing by the CD8+ T cells elicited by the killing agent. The ART drugs reduce or prevent virus spread to uninfected cells. Similarly, use of other shocking and killing agents described herein alone or in combination provide alternate embodiments of this method.

In one aspect, the present invention is a method of reducing or eliminating viral reservoirs in an individual in need thereof by:

(i) administering one or more shock agents, and

(ii) administering one or more kill agents, thereby reducing reduce the viral reservoirs.

In exemplary embodiments, step (i) occurs before step (ii), i.e., the shock agent is administered before the kill agents.

In exemplary embodiments, the shock and kill agents are administered simultaneously or on the same day, i.e., within the same 24 hour period.

In one embodiment, the kill agent in step (ii) is administered between about 1 and about 4 weeks, between about 1 and about 2 weeks, or about one week after the shock agent is administered in step (i).

In one embodiment, the kill agent in step (ii) is administered between about 1 to about 10 days, about 1 to about 7 days, about 1 to about 5 days, about 1 to about 3 days, about 3 to about 7 days, about 5 to about 6 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days after

administration of shock agent in step (i).

In another embodiment, the shock agent is administered about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, or about 24 hours before the kill agent.

In exemplary embodiments, the reduction of the viral reservoirs is complete, i.e., the virus is eradicated or eliminated.

In another embodiment, the method further comprises the step of: (iii) measuring the presence of viral reservoirs for reduction.

In one embodiment, viral reservoirs are measured at about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 weeks after step (ii). In another embodiment, viral reservoirs are measured at about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months. In a further embodiment, viral reservoirs are measured about 1, about 2, about 3, about 4, or about 5 years after step (ii).

In one embodiment, viral load is determined after administration of one or more shock agents as an indicator of reservoir activation. According to this embodiment, the present invention is a method of reducing or eliminating viral reservoirs in a subject in need thereof, comprising:

(i) administering one or more shock agents;

(ii) determining viral load; (iii) administering one or more kill agents at peak viral load.

Viral load may be determined, for example, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours or about 24 hours after administration of the shock agent. In other embodiments, viral load may be determined about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days after administration of the shock agent. In still further embodiments, viral load may be determined about 1, about 2, about 3, about 4, about 5, about 6 or about 7 weeks or more after administration of the shock agent.

In one embodiment, viral load is determined in a clinical setting, i.e., by a medical professional or support staff. In another embodiment, viral load is determined by the individual, and when peak viral load is detected the individual notifies the healthcare provider to obtain administration of the killing agent. Methods and kits for determining viral load by an individual are known in the art.

In one embodiment, the peak viral load occurs between about 2 and about 4 hours, about 4 and about 6 hours, about 6 and about 8 hours, about 8 and about 10 hours, about 10 and about 12 hours, about 12 and about 14 hour, about 14 and about 16 hours, about 16 and about 18 hours, about 18 and about 20 hours, about 20 and about 22 hours, or about 24 hours.

In another embodiment, the peak viral load occurs between about 1 and about 4 weeks, about 1 and about 2 weeks, or about 1 week after administration of the shock agent.

In one embodiment, the peak viral load occurs between about 1 and about 7 days, about 1 and about 5 days, or about 1 and about 3 days.

In one embodiment, the peak viral load occurs between about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In another embodiment, the kill agent is administered at about the time of peak reservoir activation as measured by viral load. In exemplary embodiments, the kill agents is administered within about 1, about 2, about 4, about 6, about 8, about 12, about 18, about 20 or about 24 hours of peak viral load. In one embodiment, the kill agent in step (iii) is administered about 1-10 day, 1-7 day, 1-

5 day, 1-3 day, about 1 day, about 2 day, about 3 day, about 4 day, about 5 day, about 6 day, or about 7 day after administration of shock agent in step (i) or determination of peak viral load in (ii).

In other embodiments, the kill agent in step (iii) is administered to the subject about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, about 20 weeks, or about 24 weeks after administration of shock agent in step (i) or determination of peak viral load in (ii).

In another embodiment, the method further comprises the step of:

(iv) measuring the presence of viral reservoirs for reduction. In one embodiment, reservoirs are measured in (iv) at about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8 weeks after step (iii). In another embodiment, reservoirs are measured in step (v) about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 months after step (iii). In further embodiments, reservoirs in (iv) are measured at about 1, about 2, about 3, about 4, or about 5 years after step (iii). In one embodiment, the individual is undergoing antiretroviral therapy prior to, during, and after administration of shock and kill agents.

In one embodiment, the antiretroviral agent is administered prior to and during the administration of shock and kill agents.

In one embodiment, in addition to flushing viral reservoirs, the shock agent is also capable of priming an immune response. The immune response is biased towards CD4+ T cells, and contributes to control by providing CD4+ T cell help for the CD8+ T cell response. Without being bound by any particular theory, is believed that immunostimulatory effects of a vaccine shock agent work in conjunction with, and may increase the potency of, kill agent-stimulated immune responses and ART to reduce or eradicate viral reservoirs. In another aspect, the present methods also provide multiple cycles of shock/kill agent administration to be repeated at regular intervals for continued reduction of the viral reservoir or until such time that viral reservoirs are eliminated.

Additional doses of one or more of the shock agents described herein and/or the kill agents described herein may be administered to a subject. Repeating a shock/kill administration cycle provides a protocol that is repeated at regular intervals for continued flushing/eradication of the reservoir.

In one aspect, the present invention is a method of reducing or eliminating viral reservoirs in an individual in need by:

(i) administering one or more shock agents

(ii) administering one or more kill agents,

(iii) repeating steps (i) and (ii) to reduce or eliminate viral reservoirs.

Where the method is repeated, the term "cycle" is used to refer to steps (i) and (ii), collectively.

In another embodiment, the method further comprises the step of:

(iv) measuring the presence of viral reservoirs for reduction, and

(v) repeating steps (i) and (ii) if further reduction in viral reservoirs is required.

In exemplary embodiments, the shock/kill cycle is repeated two or more times. In a particular embodiment, the shock/kill cycle is repeated two or more times at regular intervals.

In one embodiment, reservoirs are measured at about 1, 2, 3, 4, 5, 6, 7, or 8 weeks, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1, 2, 3, 4, or 5 years after step (ii).

In one embodiment, the method is repeated at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, or more than 8 times. Put another way, treatment involves at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or more than 8 cycles.

In one embodiment, about 2-8, about 4-8, or about 6-8 shock/kill cycles are provided. In one embodiment, about 1-4-week, 2-4 week, 3-4 week, 1 week, 2 week, 3 week, 4 week or more than 4 week intervals are provided between cycles.

In one specific embodiment, 4-week intervals are used between cycles.

In one embodiment, treatment is continued until such time that reservoirs are reduced or eliminated as measured by methods known in the art to assess viral reservoir levels.

In one embodiment, the reduction or elimination of reservoirs is measured by TILDA as compared to reservoir levels prior to treatment.

In one embodiment, the reduction or elimination of reservoirs is measured by integrated DNA test as compared to reservoir levels prior to treatment. In one embodiment, reduction of viral reservoir levels is about 2 fold as compared to levels prior to treatment. In another embodiment, reduction of viral reservoir levels is about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, or more than 7 fold as compared to levels prior to treatment.

In one embodiment, viral reservoir levels are undetectable after shock/kill treatment. In another aspect, other infectious diseases that need to flush latent reservoirs might be managed or controlled through this method of simultaneous administration of vaccine shock/kill agents containing the corresponding genetic material for infectious diseases other than HIV with or without appropriate concomitant drug therapies.

B. Administration

The compositions described herein can be administered in a variety of ways including through any parenteral or topical route. For example, shock and kill agents may be administered by intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular methods.

Inoculation can be, for example, with a hypodermic needle, needleless delivery devices such as those that propel a stream of liquid into the target site, or with the use of a gene gun that bombards DNA on gold beads into the target site. The shock and kill agents can be administered to a mucosal surface by a variety of methods including, but not limited to, electroporation, intranasal administration (e.g., nose drops or inhalants), or intrarectal or intravaginal administration by solutions, gels, foams, or suppositories.

Alternatively, shock and kill agents can be orally administered in the form of a tablet, capsule, chewable tablet, syrup, emulsion, or the like. In an alternate embodiment, agents can be administered transdermally, by passive skin patches, iontophoretic means, and the like.

Any physiologically acceptable medium can be used to administer a shock or kill agent to a subject. For example, suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. The media may include auxiliary agents such as diluents, stabilizers (i.e., sugars (glucose and dextrose were noted previously) and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, additives that enhance viscosity or syringability, colors, and the like.

Preferably, the medium or carrier will not produce adverse effects, or will only produce adverse effects that are far outweighed by the benefit conveyed.

A therapeutically effective amount of a vector (whether considered the first, second, third, etc. vector) can be administered by an intramuscular, a mucosal, or an intradermal route, together with a physiologically acceptable carrier, diluent, or excipient, and, optionally, an adjuvant. A therapeutically effective amount of the same or a different vector can subsequently be administered by an intramuscular or an intradermal route, together with a physiologically acceptable carrier, diluent, or excipient, and, optionally, an adjuvant to boost an immune response. Such components can be readily selected by one of ordinary skill in the art, regardless of the precise nature of the antigens incorporated in the vaccine or the vector by which they are delivered.

DNA can be delivered in a variety of ways, any of which can be used to deliver the plasmids of the present disclosure to a subject. For example, DNA can be injected in, for example, saline (e.g., using a hypodermic needle) delivered as a ballistic (by, for example, a gene gun that accelerates DNA-coated beads) or delivered by electroporation. Saline injections deliver DNA into extracellular spaces, whereas gene gun deliveries bombard DNA directly into cells. Electroporations transiently disrupt the integrity of cellular membranes, thereby allowing entry of the DNA. The saline injections require much larger amounts of DNA (typically 100-1000 times more) than the gene gun (Fynan et al, Proc. Natl. Acad. Sci. U.S.A. 90: 11478-11482, 1993). These types of delivery also differ in that saline injections and electroporations bias responses towards type 1 T-cell help, whereas gene gun deliveries bias responses towards type 2 T-cell help (Feltquate et al, J. Immunol. 158:2278-2284, 1997; Pertmer et al, J. Virol. 70:6119- 6125, 1996). DNAs injected in saline rapidly spread throughout the body. DNAs delivered by the gun are more localized at the target site.

Following either method of inoculation, extracellular plasmid DNA has a short half-life of about 10 minutes (Kawabata et al., Pharm. Res. 12:825-830, 1995; Lew et al., Hum. Gene Ther. 6:553, 1995). Vaccination by saline injections can be intramuscular (i.m.), intradermal (i.d.), or mucosal (as described below in more detail); gene gun deliveries can be administered to the skin or to surgically exposed tissue such as muscle.

While other routes of delivery are generally less favored, they can nevertheless be used to administer the compositions of the disclosure. For example, the DNA can be applied to the mucosa or by a parenteral route of inoculation. Intranasal administration of DNA in saline has met with both good (Asakura et al, Scand. J. Immunol. 46:326-330, 1997; Sasaki et al, Infect. Immun. 66:823-826, 1998b) and limited (Fynan et al, Proc. Natl. Acad. Sci. U.S.A. 90: 11478- 82, 1993) success. The gene gun has successfully raised IgG following the delivery of DNA to the vaginal mucosa (Livingston et al, Ann. New York Acad. Sci. 772:265-267 ', 1995). Some success at delivering DNA to mucosal surfaces has also been achieved using liposomes

(McCluskie et al, Antisense Nucleic Acid Drug Dev. 8:401-414, 1998), microspheres (Chen et al, J. Virol. 72:5 ^* 5 ^* -57 ^* 61, 1998a; Jones et al, Vaccine 15:814-817, 1997), and recombinant Shigella vectors (Sizemore et al, Science 270:299-302, 1995; Sizemore et al, Vaccine 15:804- 807, 1997). Agents such as these (liposomes, microspheres, and recombinant Shigella vectors) can be used to deliver the nucleic acids of the present disclosure.

C. Dosing The dose of DNA needed to raise a response depends upon the method of delivery, the host, the vector, and the encoded antigen. The method of delivery may be the most influential parameter. From 10 μg to 5 mg of DNA is generally used for saline injections of DNA, whereas from 0.2 μg to 20 μg of DNA is used more typically for gene gun deliveries of DNA. In general, lower doses of DNA are used in mice (10-100 μg for saline injections and 0.2 μg to 2 μg for gene gun deliveries), and higher doses in primates (100 μg to 1 mg for saline injections and 2 μg to 20 μg for gene gun deliveries). The much lower amount of DNA required for gene gun deliveries reflect the gold beads directly delivering DNA into cells.

D. Reservoir Assay

In one embodiment, the method includes a step of measuring the presence of reservoirs after shock/kill treatment.

Metrics of viral infection are assayed to determine baseline infection and/or reservoir levels as well as effectiveness of treatment. Examples of metrics of viral infection that can be assayed include, but are not limited to viral reservoirs, viral RNA and T Cell levels. Suitable assays to measure viral reservoirs include, but are not limited to viral outgrowth assay, limiting dilution co-culture, measurement of total cell-associated HIV DNA by PCR, quantification of test for integrated DNA by Alu-gag PCR, quantification of 2-LTR circles by PCR, and TILDA assay although other assays for measuring viral reservoirs are known to those in the art.

Other methods of measuring viral reservoirs are known and under development. One such method is co-cultivation of stimulated cells (PBMC) with sensitive indicator cells. (See Bullen et al, Nat Med. 2014 April ; 20(4): 425-429).

A PCR test for integrated DNA is used to measure the number of HIV DNA copies per million CD4+ T cells. CD4+ T cells are isolated from patients. The samples are subjected to quantitative PCR analysis, in which a specific sequence indicative of the presence of HIV genome was amplified. The number of HIV genomes per sample is derived from the quantitative PCR results using statistical methods known to those in the art.

The TILDA (Tat/rev Induced Limiting Dilution Assay) is a relatively new method to quantify the latent HIV reservoir. While integrated DNA tests quantify the number of HIV genomes (including both replication-competent genomes and defective genomes), TILDA measures HIV RNA that has been multiply spliced, meaning that it has gone through the first few steps in HIV replication. Detection of multiply-spliced RNA indicates the presence of HIV genome that, upon activation, is capable of producing RNA with correct splicing sites. In comparison to integrated DNA tests, TILDA is a more accurate indicator of the infectious potential of an HIV-infected individual's reservoir. In the TILDA assay, CD4+ T cells are isolated from the body and stimulated for 12 hours with PMA and ionomycin, a combination of drugs known to be extremely potent in activating latent HIV. The samples of stimulated cells, now containing activated virus, are serially diluted. Multiple dilutions of each sample are then tested for multiply-spliced HIV-1 tatrev RNA

(msHIV-tatrev RNA) by quantitative RT-PCR. Using a maximum likelihood statistical method, the frequency of cells with inducible msHIV RNA is derived from the quantitative PCR results. TILDA is highly precise, sensitive, and reproducible. It is also much faster than many other techniques and does not require specialized equipment beyond common laboratory instruments.

Assays are performed before administration of vaccine and at a reasonable time after administration of vaccine. In one embodiment, these criteria are assayed at about 5 days, about 7 days, about 9 days, about 10 days, about 12 days, about 14 days, about 18 days, about 21 days, or about 24 days after administration of DNA or MVA. In one embodiment, the viral RNA is measured 7 days after vaccine administration. In another embodiment, viral RNA and T cells are measured 14 days after vaccine administration.

In one embodiment, measurements of viral infection are assayed before vaccine administration to determine baseline values. Measurements are repeated after treatment to assess effectiveness of the treating.

All patents and patent publications referred to herein are hereby incorporated by reference.

EXAMPLES

EXAMPLE 1 : CpG ODN, but not GpC ODN or GEO-D03, activated pro viral expression in ACH-2 cells.

A prior study has shown that unmethylated CpG motifs can activate proviral DNA in the ACH-2 cell line, which harbors a latent provirus. Unmethylated CpG motifs are recognized as activators of a number of cellular signaling pathways, which include pathways such as NFKB and NFAT-1 which affect provirus expression. Certain types of molecules, including cytokines and histone deacetylase (HDAC) inhibitors, are capable of inducing activation of latent proviral HIV- 1. ( Chun TW et al, J Exp Med (1998), 188:83-91; Rasmussen TA et al, Hum Vacc

Immunother (2013), 9:993-1001)

As described herein, activation of latent HIV-1 is desired for the "shock" step of any "shock and kill" treatment. Based on a hypothesis that CpG ODN could have an effect similar to that seen with cytokines and HDAC inhibitors, Scheller et al. tested the effect of CpG ODN on ACH-2 cells, which are latently infected with HIV-1. The results clearly demonstrated that CpG ODN were capable of activating latent HIV-1 in ACH-2. (Scheller C et al, J Biol Chem (2004), 279:21897-21902.)

Using a similar approach, CpG ODN, GpC ODN (similar to CpG ODN but without immunostimulatory CpG motifs) and GEO-D03 DNA were tested on ACH-2 cells. The ACH-2 cells were cultured under standard conditions and then exposed to CpG ODN, GpC ODN and GEO-D03.

ACH-2 cells were cultured at 37°C in 5% C02 atmosphere in RPMI-1640 (Corning), containing 10% fetal bovine serum, penicillin, and streptomycin. Cells were incubated in a 24 well plate with stimulants diluted in DNA resuspension buffer (DNA RB; 25mM sodium phosphate, 0.2mM EDTA, 120mM sodium chloride, and l%v/v ethanol). Stimulants tested were CpG ODN 2006 (Invivogen), negative control ODN 2137 (Invivogen), DNA vaccine GEO-D03 (GeoVax), DNA RB only or medium only for 20 hours. Cells were harvested and then stained with an antibody specific for HIV-1 Gag protein. The level of Gag expression, which indicates the degree of HIV-1 activation, was quantified by flow cytometry of the stained cells.

As shown in FIG. 1, the level of HIV-1 activation increased upon stimulation with CpG ODN, whereas GpC ODN and GEO-D03 were not effective at stimulating HIV-1 activation. This suggests that GEO-D02 DNA might not necessarily reactivate latent proviral DNA by virtue of its containing CpG motifs.

EXAMPLE 2: Transient increases in viral RNA were associated with the administration of GEO- D02 DNA (pGA2/JS7) to infected and drug-treated humans.

In spite of the inactivity of GEO-D03 vaccine to stimulate HIV activation in ACH-2 cells, a recently-completed open-label clinical trial (GV-TH-01) of the GEO-D02 DNA vaccine and the MVA62B recombinant viral vaccine in nine HIV- 1 -infected, ART-suppressed patients demonstrated stimulation of HIV activation in human subjects. The purpose of the trial was to test the GEO-D02/MVA62B prime-boost regimen for therapy of HIV- 1.

All participants in the trial were infected with HIV, and all had initiated Anti-Retroviral Therapy (ART) within 18 months of documented seroconversion. The participants were vaccinated with the GEO-D02 DNA vaccine at 0 and 8 weeks, and then with the MVA62B viral vaccine at 16 and 24 weeks. At 8 weeks after the final MVA inoculation, a treatment interruption was initiated. Prior to treatment interruption, any patients taking Efavirenz underwent a two-to-six week washout for effavirenz in the presence of a drug regimen in which all agents had similar half lives. ART treatment was interrupted for 12 weeks, and then participants were returned to ART and monitored for an additional 24 weeks.

Throughout the trial, participants were tested at scheduled intervals for HIV-1 viral load. Of the nine individuals who received the vaccines, four showed transient but measurable increases in viral load that occurred within one week after receipt of the GEO-D02 DNA vaccine . In all there were five examples of these transient increases ("blips"). Participants 01-6, 01-8 and 01-9 each experienced a single blip within one week of a DNA inoculation. Participant 01-5 experienced two blips within one week of each of the two DNA inoculations.

Other transient increases occurred that were not immediately associated with DNA inoculations. Participant 01-3 experienced several blips, with no apparent association with timing of vaccinations. Participant 01-7 experienced two blips, each coinciding with (not following) a vaccination. Participant 01-8, whose viral load rose transiently after the first DNA vaccination, experienced a second blip coinciding with (not following) the second DNA vaccination. FIG. 2 illustrates transient increases in viral load observed during the immunization phase of the trial. Those that appeared to be stimulated by DNA inoculations are indicated with green arrows, and the corresponding participant IDs are circled in green.

EXAMPLE 3 : Administration Regime to Reduce or Clear HIV from Viral Reservoirs in Patients Infected with HIV

A regimen using DNA as a shock and an MVA HIV vaccine as a kill in participants on continuous antiretro viral therapy is shown in Figure 4. The administrations are timed to have the shock effect and reactivation of latent virus coincide with the kill effect of elicited CD8+ responses. Administration of vector in the presence of systemic anti-retro viral therapy prevents or reduces spread of infection to uninfected cells.

HIV DNA and MVA62B vectors are administered at 0, 4, 8, and 12 weeks in the presence of antiretro viral therapy. Viral RNA and T cell response are assayed 7 or 14 days after administration of vectors. Reservoirs are assayed at 0 weeks, and 20, 28 and 36 weeks to determine effectiveness of treatment.

EXAMPLE 4: An example of the reduction of a viral reservoir of an individual who underwent spontaneous expression of virus at the time of a MVA vaccine response.

A spontaneous transient enhancement of viral RNA occurred coincident with the 2 ^nd MVA administration in in GV-TH-01 in participant 01-7 (see FIG. 2). In this participant, the transient reactivation of virus (evidenced by the transient presence of viral RNA in plasma) occurred when a substantial CD8+ T cell response had been raised. This patient underwent significant decreases in both proviral DNA in CD4+ T cells and the expression of spliced Tat and Rev mRNA from latent proviral DNA (TILDA assay- Tat-Rev induced Limiting Dilution Assay) (Figure 4). These results suggest that shock and kill protocols in drug-treated patients that cause transient increases in viral RNA at the same time that effector CD8+ T cells are present can lead to reservoir reductions.

Participants' viral reservoirs were measured by two methods. Proviral DNA in CD4+ T cells was measured by a standard integrated DNA test. In addition, spliced tat and rev mRNA from latent proviral DNA were quantified using the Tat-Rev Induced Limiting Dilution Assay (TILDA). In the TILDA test, CD4+ T cells are stimulated with PMA and ionomycin, potent activators of latent HIV. Then, an RT-PCR method is employed to quantify multiply-spliced HIV RNA. Unlike the standard quantitative PCR, which detects both intact and defective HIV-1 genomes, TILDA quantifies only intact virus.

All patents and publications referenced herein are hereby incorporated by reference in their entirety.

Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.