FIELD OF THE INV ENTION

The present invention relates to vaccine production. More specifically the present invention relates to the production of genetically engineered membrane-associated particulate subunit vaccine for Zika virus, a mosquito-borne flavivirus.

BACKGROUND OF THE INVENTION

Zika virus (ZIKV) is a flavivirus, phylogenetically related to West Nile virus (WNV), Yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and the four antigenically distinct serotypes of dengue viruses (DENV-1, DENV-2, DENV-3 and DENV-4) (Pierson TC, Diamond MS. Flaviviruses. In: Knipe DM, Howley PM, editors-in-chief. Fields Virology, 6e. Philadelphia: Wolters Kluwer and Lippincott Williams & Wilkins; 2013. p. 747-794). It has a plus sense genomic RNA of ~11 kilobaseswhich contains a single open reading frame encoding three structural proteins, capsid, pre-membrane (prM) and envelope (E), and seven non- structural proteins, 1 (NS1), NS2a, NS2b, NS3, NS4a, NS4b and NS5(Figure 1A) (Pierson TC, Diamond MS. The emergence of Zika virus and its new clinical syndromes. Nature. 2018; 560: 573-581 ). ZIKV is spread to humans by Aedes mosquitoes andhas been historically associated with mild and self-limiting fever akin to dengue fever (Gatherer D, Kohl A. Zika virus: a previously slow pandemic spreads rapidly through the Americas. J Gen Virol. 2016; 97: 269-273; Lazear HM, Diamond MS. Zika virus: New clinical syndromes and its emergence in the Western hemisphere. J Virol. 2016; 90: 4864-4875). ZIKV may also be transmitted between humans sexually (Musso D, et. al. Potential sexual transmission of Zika virus. Emerg Infect Dis. 2015; 21: 359-361 ) or vertically (Besnard M, et al, Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill. 2014; 19(13) :pii 2075 /; M 1 ak ar J, et.al. Zika virus associated with microcephaly. N Engl J Med. 2016; 374: 951-958). ZIKV outbreaks have been documented in recent times starting from 2007 until about mid-2016, by which time it had spread to several countries in the Pacific as well as to countries in Asia, Africa and the Americas (Gatherer D, Kohl A. Zika virus: a previously slow pandemic spreads rapidly through the Americas. J Gen Virol. 2016; 97: 269-273; Baud D, et ah, An update on Zika virus infection. Lancet. 2017; 390: 2099-2109; Lessler J, et. al. Assessing the global threat from Zika virus. Science. 2016; 353: aaf8160). More severe clinical manifestations of ZIKV infection became apparent in the Brazilian outbreaks during 2015. Exposure of fetuses to ZIKV in the first trimester of pregnancy has been linked to neurodevelopmental malfunction resulting in congenital birth defects including microcephaly (Mlakar J, et. al, Zika virus associated with microcephaly. N Engl J Med. 2016; 374: 951 -95 S; Ras ussen et al, Zika virus and birth defects-reviewing the evidence for causality. N Engl J Med. 2016; 374: 1981-1987; Rubin et al, Zika virus and microcephaly. N Engl J Med. 2016; 374: 984-985; CalvetG et al, Detection and sequencing of Zika virus from amniotic fluid of fetuses with microcephaly in Brazil: a case study. Lancet Infect Dis. 2016; 16: 653-660). On the other hand, ZIKV infection in adults appears to lead to an autoimmune disease known as Guillain Barre syndrome (GBS) (Cao-LormeauVM et al, Guillain-Barre syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet 2016; 387: 1531- 1539). These neurological abnormalities have been linked to the capacity of ZIKV to infect human neural progenitor cells (Tang H et al, Zika virus infects human cortical neural progenitors and attenuates their growth. Cell Stem Cell. 2016; 18: 587-590). ZIKV infections are beginning to be reported in India. It is currently estimated that more than 2 billion people live in areas considered suitable for ZIKV transmission (Messina E et al, Mapping global environmental suitability for Zika virus. eLife. 2016; 5: el 5272).

ZIKV vaccine development is complicated by the existence of antibody-dependent enhancement (ADE) phenomenon, stemming from the interaction between ZIKV on the one hand and DENVs on the other. Not only do these viruses share the same mosquito vector (WeavenSV et al, Zika virus: History, emergence, biology, and prospects for control. Antiviral Res. 2016; 130: 69-80), but also are genetically and antigenically similar. In vitro data show that antibodies induced by ZIKV can cross-react with and enhance infection by DENVs and vice versa (Culshaw et al, The immunopathology of dengue and Zika virus infections. Current Opinlmmunol 2017; 48: 1-6; Harrison.VC. Immunogenic cross-talk between dengue and Zika viruses. Nature Immunol. 2016; 17: 1010-1012.). Given the high sero-prevalence of DENV in areas that witnessed recent ZIKV epidemics (Brathwaite/J/c : 0 et al, Review: The history of dengue outbreaks in the Americas. Am J Trop Med Hyg. 2012; 87: 584-593; CastanhaPMS' et al, Force of infection of dengue serotypes in a population -based study in northeast razil. Epidemiol Infect. 2013; 141: 1080-1088; Braga et al, Seroprevalence and risk factors for dengue infection in socio-economically distinct areas of Recife, Brazil. Acta Trop. 2010; 113: 234-240 ), it has been hypothesized that pre-existing DENV antibodies may have mediated ADE of ZIKV (DejnirattisaiWe/ al, Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat Immunol. 2016; 17: 1102-1108; BardinaSV et al, Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity. Science. 2017; 356: 175-180). As a corollary to this, the reverse could be an equally likely possibility. This draws support from recent experiments which have demonstrated that ZIKV envelope domain Ell-specific monoclonal antibodies (mAbs) from ZIKV-infected patients can enhance DENV-2 infection both in vitro and in vivo (Stettler et al, Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826). Therefore, deployment of a flaviviral vaccine is complex as it must ensure that the risk of enhancing a related flaviviral infection must be avoided. Thus, for a safe ZIKV vaccine, a key attribute is that it must not contribute to ADE of DENV infection.

The ZIKV E protein is the major component involved in receptor binding, membrane fusion and in recognition by the host immune system and contains epitopes recognized by potent murine (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027) and human (Stettler K et al, Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826; Wang Q et al, Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus. SciTransl Med. 2016; 8: 369ral79) neutralizing antibodies (nAbs). Just like its other flaviviral counterparts, the ZIKV E protein is also organized into three distinct domains, referred to as envelope domain I (EDI), EDII and EDIII {Dai L et al, Structures of the Zika virus envelope protein and its complex with a flavivirus broadly protective antibody. Cell Host Microb. 2016; 19: 696-704). On the other hand, the flaviviralprM protein appears to function as a chaperone of the E protein, preventing premature fusion of the immature virion as it transits the trans-Golgi network within the infected cell. Consistent with this,cryo-EM analyses reveal that the overall structure of the mature ZIKV particle is similar to that of other flaviviruses (SirohiDe/ al, The 3.8 A resolution cryo-EM structure of Zika virus. Science. 2016; 352: 467-470; Kostyuchenko VA et al, Structure of the thermally stable Zika virus. Nature. 2016; 533: 425-428). Comparative sequence analysis of ancestral and contemporary ZIKV strains has led to the identification of a single mutation in the ZIKV prM protein (S139N) (Yuan L et al, A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. Science. 2017; 358: 933-936). In the light of neurodevelopmental defects associated with recent ZIKV outbreaks, this observation appears to link prM to the increased neurovirulence of the latter strains and has led to the hypothesis that the S139N prMmutation presumably confers tropism for neural progenitor cells (ScreatonG&Mongkolsapaya J, Evolution of neurovirulentZika virus. Science. 2017; 358: 863-864). However, unlike for ZIKV E, the nature of the human immune response to ZIKV prM is not known at present, making it difficult to gauge the possible utility of including prM in ZIKV vaccines.

Numerous ZIKV vaccine candidates, employing multiple technologies and platforms, are being explored by various investigators. These include live attenuated and inactivated vaccines as well as those based on recombinant virus vectors, nucleic acids, proteins and peptides ( Abbink P et al, Zika virus vaccines. Nature. 2018; 16: 594-600; Poland GA et al, Development of vaccines against Zika virus. Lancet Infect Dis. 2018; 18: e211-219; Wilder-Smith et al, Zika vaccines and therapeutics: landscape analysis and challenges ahead. BMC Medicine. 2018; 16:84). Several of these, including an mRNA-based vaccine, a recombinant measles virus-vectored vaccine and several plasmid DNA-based and inactivated ZIKV candidates are in phase 1 trials with the mRNA and the measles virus-vectored vaccines in phase 2 {Abbink P et al, Zika virus vaccines. Nature. 2018; 16: 594-600; Barrett, Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation. npj Vaccines. 2018; 3: 2-/;RichnerJM& Diamond MS, Zika virus vaccines: immune response, current status, and future challenges. Current Opinlmmunol. 2018; 53: 130-136 ; Wilder-Smith et al, Zika vaccines and therapeutics: landscape analysis and challenges ahead. BMC Medicine. 2018; 16:84; WHO Vac Pipeline Tracker)(

https ://docs. google . com/ spreadsheets/ d / 19otvIN cay JURCMg76xW 04Kvuyed YbMZDcX qbyJGdcZM/pubhtml#.). Apart from the live attenuated vaccines and purified inactivated vaccines, all nucleic acid-based and most protein-based vaccine candidates are designed to encode the two ZIKV structural proteins prM and E (Barrett, Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation npj Vaccines. 2018; 3: 24; Richner& Diamond, Zika virus vaccines: immune response, current status, and future challenges. Current Opinlmmunol. 2018; 53: 130-136; WHO Vaccine Pipeline Tracker)

(https ://docs .googl e. com/ spreadsheets/ d / 19otvIN cayJURCMg76xW 04Kvuyed YbMZDc XqbyJGdcZM/pubhtml#.). Thus far, none of these ZIKV vaccines are designed to address the issue of eliminating ADE of DENVs.

US2018340181 discloses compositions including a virus-like particle (VLP)-based vaccine displaying a portion of ZIKV envelope protein (E) domain III (Dill) and a portion of ZIKV envelope protein (E) and related methods are disclosed herein. Further, compositions including vaccines comprising a portion of ZIKA virus E protein, wherein the portion of ZIKA virus E protein is either a full-length version of ZIKA virus E protein or a functionally equivalent version of the full-length ZIKA virus E protein, are disclosed. US’ 181 mentions that ZIKV E (zE) is a major target of host antibody responses and its EDIII (zDIII) has been found to be targeted by several ZIKV-specific antibodies with strong neutralizing activities. US’ 181 further mentions“Since ZIKV and DENV are closely related and co-circulate geographically, ZIKV vaccines that are based on common epitopes of the two viruses may elicit cross-reactive antibodies that augment infection of DENV in vaccinated subjects when they are exposed to DENV secondarily. This hypothesis is supported by the finding that cross-reactive antibodies targeting the highly conserved fusion loop in EDII (EDII-FL) of zE generated during natural ZIKV infection can enhanced DENV infection both in cell culture and in mice. Therefore, vaccine strategies based on antigens that can avoid induction of cross-reactive antibodies should minimize the risk of ADE of DENV infections.” US2018340181 describes the use of the plant expression system (N. benthamiand) to produce two different vaccine immunogens: (i) zEDIII displayed on HBV core VLPs and (ii) zE with mutated fusion loop (zE-FL- mutant). This mutation is for the purpose of avoiding the induction of cross-reactive antibodies which are implicated in ADE. The zEDIII here requires HBV core VLP as carrier for its display. US‘181 uses 50 pg VLPs per dose, which according to the inventors is equivalent to 50 pg zEDIIEdose. The purification of the vaccine of US‘181 involves centrifugation on a sucrose gradient, which is undesirable in terms of scale-up of purification.

CN108503696 teaches a subunit Zika virus vaccine expressed by yeast cells. Specifically, the subunit Zika virus vaccine developed by using yeast cells in the invention has the advantages of high yield, high purity, good stability and easy purification; meanwhile, because the subunit Zika virus vaccine contains no viral nucleic acid component, the subunit Zika virus vaccine is free of the possibility of mutation restoration and has high safety. CN108503697 discloses similar invention where the expression host is Drosophila. These two inventions concern the production of ZIKV EDIII and ZIKV E80 proteins as vaccine antigens using the yeast expression system (CN ‘696) or the Drosophila insect cell expression system (CN‘697). In both systems, the two vaccine antigens are designed to be secreted into the culture supernatant, using either the a-factor secretion signal (CN‘696) or the Drosophila BiP secretion signal (CN’697). The CN’696 vaccine is a monomer and the CN’697 vaccine is a dimer and these vaccines do not form higher ordered structures. In terms of neutralizing antibody titers elicited the yeast- expressed ZIKV EDIII and insect cell-expressed ZIKV E80 appear to be the preferred vaccine candidates, however the two vaccines have not been tested for ADE.

Thus there is a need to design a ZIKV vaccine which specifically address the issue of eliminating ADE of DENVs.

OBJECTS OF THE INVENTION

It is an object of the present invention to produce ZIKV prM-lacking, membrane- associated particle (MAP) form of recombinant ZIKV E protein. It is another object of the present invention to provide a method for efficient recombinant expression, retrieval and refolding of the ZIKV E protein.

It is further object of the present invention to provide a method of expression, retrieval and refolding of ZIKV E in a way that permits its self-assembly into particulate form with free surface accessibility of ZIKV E domain III (ED III), endowing it with the capacity to induce specific ZIKV-neutralizing, but flavivirus-non-enhancing antibodies.

It is yet another object of the present invention to provide a safe, efficacious and inexpensive ZIKV vaccine.

It is also another object of the present invention to provide a means (kit or method) of detecting ZIKV infection.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention there is provided a subunit Zika virus (ZIKV) vaccine comprising: a membrane-associated particulate form of immunogen rec ZIKV envelope (E) protein, wherein said rec ZIKV E protein comprises of domains I, II and III and said domain III is placed on the membrane surface and is freely accessible to ZIKV EDIII-specific antibodies.

According to another embodiment of the present invention there is provided a method of producing the ZIKV subunit vaccine as claimed in any of claims 1-9, comprising the steps of :

i. introducing a DNA encoding the vaccine immunogen into a host cell,

ii. culturing said host cell,

iii. inducing expression, and

iv. retrieving and refolding the subunit vaccine in purified particulate form.

According to a further embodiment of the present invention there is provided a kit comprising a membrane-associated particulate form of immunogen rec ZIKV envelope (E) protein, said rec ZIKV E protein comprises of domains I, II and III and said domain III is freely accessible to ZIKV EDIII-specific antibodies, wherein said kit is capable of detecting ZIKV-specific antibodies in biofluids such as blood, plasma, serum, urine and saliva.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

Figure 1 illustrates the recombinantZIKV E antigen in accordance with the present invention.

Figure 2 illustrates the comparison of ZIKV and DENV E proteins.

Figure 3 illustrates the expression screening of transformants in accordance with the present invention.

Figure 4 illustrates the localization and purification of rec ZIKV E protein.

Figure 5 illustrates thephysical characterization of purified rec ZIKV E protein.

Figure 6 illustrates the alum formulation of recombinant ZIKV E MAPs and the immunization schedules.

Figure 7 illustrates the immunological evaluation of rec ZIKV E MAP -induced antibodies.

Figure 8 illustrates the evaluation of DENV enhancement by BALB/c anti-rec ZIKV E MAP antiserum/// vitro.

Figure 9 illustrates the evaluation of the enhancement potential of anti-rec ZIKV E MAP antibodies in vivo.

Figure 10 illustrates the in vivo ADE model utilized to assess the ZIKV-enhancement potential of anti-rec ZIKV E MAP antibodies.

PET ATT, ED DESCRIPTION OF THE INVENTION

The present invention provides a highly immunogenic ZIKV prM-lacking recombinant membrane-associated particulate subunit vaccine comprising the recombinant ZIKV E protein, comprising envelope domains I, II and III, and surface-displaying ZIKV EDIIF This vaccine, by virtue of displaying ZIKV EDIII is endowed with the capacity to elicit an immune response in mammals, which is capable of specifically neutralizing ZIKV infection of susceptible mammalian cells. Further, this membrane-associated particulate vaccine, by virtue of not containing prM and not exposing cross-reactive epitopes, does not enhance infection of susceptible cells byflaviviruses, including but not limited to, ZIKV, DENV-1, DENV-2, DENV-3 and DENV-4.

The present invention discloses a method to produce a membrane-associated Zika virus subunit vaccine using a suitable host expression system, such as the yeast Pichia pastoris. The subunit vaccine is in particulate form displaying the Zika virus envelope domain III so that it may elicit Zika virus-specific neutralizing antibodies without enhancement potential against the closely related dengue viruses.

According to one embodiment of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a signal peptide, ZIKV E protein ectodomainand an affinity tag for easy purification. This nucleic acid molecule is hereinafter referred to as ZIKV E gene (SEQ ID 1). In the sequence, nucleotide 1-6, containing the initiator codon (ATG), were introduced to facilitate cloning and expression. Nucleotides 7-108 encode the C-terminal 34 aa residues of ZIKV prM (of ZikaSPH2015). Nucleotides 109-1317 encode the first 403 aa residues of ZIKV E protein, comprising domains I, II and III (of ZikaSPH2015). Nucleotides 1318-1332 encode a pentaglycl peptide linker. Nucleotides 1333-1350 encode the 6x His tag. Nucleotides 1351-1353 (TAG) denote the stop codon. The corresponding protein encoded is referred to hereinafter as recombinant (rec) ZIKV E protein. The ectodomain of the ZIKV E protein, representing the N-terminal 80% of the full-length ZIKV E molecule, is comprised of three domains, I, II and IP (EDI, EDII and EDIII, respectively).

In a preferred embodiment of the invention, the rec ZIKV E protein may comprise the C- terminal 34 amino acid residues of ZIKV prM( signal peptide), the N-terminal 403 amino acid residues of the full-length ZIKV Eprotein (ZIKV E ectodomain) and a run of 6 histidine residues (6x His tag).

In one embodiment, the ZIKV E ectodomainmay be derived from the ZIKV strain, ZikaSPH2015. In another embodiment, the ZIKV E gene sequence may be varied to match the codon bias of the expression host.

In yet another embodiment, the ZIKV E ectodomain-encoding polynucleotide sequences of ZIKV E gene may be replaced with corresponding ectodomain-encoding polynucleotide sequences derived from any one of prevalent/available and known ZIKV strains or isolates. Examples of such ZIKV strains/isolates include, but are not limited to, ZIKA YAP 2007, H/PF/2013, BeH818995, PRVABC59, ZKV-16-097, MR766, ARB7701, ArD 128000 and IbH30656.

In another embodiment, the prM signal encoding sequences of SEQ ID 1 may be replaced by equivalent sequences from any of the other flaviviruses or by any other signal peptide known to be functional in the chosen host expression system. Examples of flaviviruses from which the signal peptide may be derived include, but are not limited to,WNV, YFV, JEV, TBEV, DENV-1, DENV-2, DENV-3 and DENV-4. Examples of other signal peptides include, but not limited to, a mating factor signal peptide of Saccharomyces cerevisiase and IgG leader peptide.

In another embodiment, the 6x His tag affinity may be replaced by any other affinity tag for ease of purification of the rec ZIKV E protein. Examples of alternative affinity purification tags include, but are not limited to, glutathione S transferase, maltose-binding protein and intein.

In yet another embodiment, the rec ZIKV E protein may be expressed without any additional affinity purification tag.

In one embodiment, the host in which the rec ZIKV E protein-encoding nucleic acid molecule is introduced, by methods known to those skilled in the art, may be any suitable prokaryotic or eukaryotic host.

In another embodiment, the ZIKV E gene may exist as an independent plasmid-borne entity in the host. In another embodiment, one or more copies of the ZIKV E gene may be integrated into the host DNA.

In another preferred embodiment, the host used may be either prokaryotic or eukaryotic, compatible with the expression of rec ZIKV E protein in a form conducive to its self- assembly into 30-50 nm particulate structures displaying ZIKV EDIII on the surface.

In a preferred embodiment, the host for rec ZIKV E expression may be a yeast host. Examples of such yeast hosts include, but are not limited to, Pichia pastoris and Hansenulapolymorpha .

In another embodiment, rec ZIKV E protein expression may be achieved under the control of any suitablesynthetic or natural, constitutive promoter.

In a preferred embodiment, rec ZIKV E protein expression may be achieved under the control of a synthetic or natural, regulated promoter.

In one embodiment, the expressed rec ZIKV E protein may be localized to the soluble fraction of host cell lysate.

In another preferred embodiment, the expressed rec ZIKV E protein may be localized to the insoluble (membrane-associated) fraction of the host cell lysate.

In one embodiment, the expressed ZIKV E protein may be purified by conventional or affinity chromatographic methods under native conditions provided the purified protein can self-assemble into ZIKV EDIII-displaying particulate form of 30-50 nm size.

In another embodiment, conventional or affinity chromatographic purification may be carried out under denaturing conditions.

In yet another embodiment, the purification may exploit any inherent affinity binding of the rec ZIKV E protein to a specific liganded matrix. In a preferred embodiment, the rec ZIKV E protein purified under denaturing conditions may be refolded in such a way as to promote self-assembly into highly immunogenic ZIKV ED Ill-displaying particulate form of 30-50 nm size.

In one embodiment, a ZIKV vaccine formulation comprising the 30-50 nm particulate rec ZIKV E protein preparation, a human use-compatible adjuvant, such as alum, and any pharmacologically acceptable ingredient may be inoculated into a mammal.

In another embodiment, the inoculation may be performed in one of multiple accepted ways, such as intradermal, intramuscular, sub-cutaneous etc.

In another embodiment, the inoculation may be performed using a range of vaccine dosages.

In yet another embodiment, the inoculation may be administered more than once and at varied intervals.

In one embodiment, the inoculation of the ZIKV vaccine formulation may be shown to elicit an appropriate ZIKV-specific immune response.

In another embodiment, the antibodies elicited by inoculation into the mammal, may be shown to be specific for unique ZIKV conformational or quaternary epitopes.

In another embodiment, these antibodies may be shown to potently neutralize ZIKV infection of susceptible mammalian cells.

In another embodiment, these antibodies may be shown to lack any ADE potential towards various ZIKV isolates as well as other flaviviruses, including but not restricted to, DENV-1, DENV-2, DENV-3 and DENV-4 in a mammal.

In yet another embodiment, the rec ZIKV E protein may be incorporated in a diagnostic test of kit to detect the presence of anti -ZIKV antibodies in patient samples as a means of identifying ZIKV infection cases. The present inventors have surprisingly found that the immunogenic ZIKV prM-lacking recombinant membrane-associated particulate subunit has higher order structuresas evident in Fig 5, panels D and E, even in the absence of prM. To achieve this, the present inventors have added the C-terminal 34 amino acid residues of prM as a signal peptide to the N-terminus of the ZIKV E ectodomain. During expression in P. pastoris , this prMsignal (which helps the recombinant protein traverse the endoplasmic reticulum and in doing so causes membrane association) is cleaved off and the resulting E ectodomain self-assembles into MAPs. Normally ZIKV virus-like particle (VLP) formation requires co-assembly of two different ZIKV proteins, prM and E. The present invention shows that higher order structures similar to VLP can be formed in the absence of prM. These have been named as‘membrane-associated particles (MAPs)’ as they have been purified from the membrane-fraction (of induced P. pastoris cells) and this also helps to distinguish them from classic VLPs, which also by definition contain prM and E. As compared to the two-protein VLPs described in the prior art, the MAPs contain only one protein.

Unique features of the present invention

(i) EDIII display: This is evident from the efficient reactivity of ZIKV EDIII- specific mAbs towards rec ZIKV E MAPs (Table 2). It is believed that the membrane-associated rec ZIKV E expressed in P. pastoris tends to self- assemble into higher order structures (based on DLS and EM analyses, Fig 5D and 5E) in such a way as to display EDIII on the MAP surface.

(ii) EDIII-directed nAb response: Ab titration experiments show that almost the entire Ab response elicited by rec ZIKV E MAPs is directed predominantly to ZIKV EDIII (Figure 7). This is consistent with the MAPs self-assembling in a manner permitting EDIII-display. Further, selective affinity depletion of EDIII-directed Abs from the immune serum correlates strongly with loss of nAb activity.

(iii) Specificity of the nAb response: Therec ZIKV E MAP -induced Abs specifically neutralized ZIKV alone potently, with absolutely no capacity to neutralize any of the four DENV serotypes (Fig 6B, table). This is quite unexpected, given the high degree of similarity (55-57%) between ZIKV and DENVs (Figure 2). Taken together with the EDIII-specific Ab depletion data it is believed that this high-degree of ZIKV specificity of the rec ZIKV E MAP -induced Ab response is because the MAPs elicit a ZIKV EDIII-focused immune response, which in turn is the outcome of the MAPs displaying EDIII effectively to the immune system.

(iv) Lack of APE against DENV: Reports in literature show that ZIKV Abs can enhance DENV infection because of the similarity between ZIKV and DENVs. Yet, the ZIKV Abs elicited by rec ZIKV E MAPs do not cause ADE of DENVs. This has been observed both in vitro (Figure 8) and in vivo (Figure 9). It is believed that the lack of DENV ADE is because of the ZIKV-specificity of rec ZIKV E MAPs-induced Abs, which are capable of neutralizing ZIKV but not DENVs. As referred to above, this ZIKV- specificity is a result of the uniqueness of the rec ZIKV E MAPs which, by display of ZIKV EDIII, elicit a ZIKV ED Ill-directed Ab response.

(v) Lack ADE against ZIKV: This is another unique feature of the present invention. In general, it is considered that a flavivirusnAb, if diluted to sub-neutralizing levels, will end up enhancing infection by the cognate flavivirusvza the Fc-receptor pathway. Surprisingly, the present inventors have found that, passive transfer of anti-rec ZIKV E MAP antiserum into riVa/2 mice (a ZIKV sensitive ADE model) to result in extremely low ZIKV nAb titers in vivo , failed to enhance a sub-lethal ZIKV challenge (Figure 10). This suggests that ZIKV EDIII-directed nAbs are inherently non-enhancing regardless of their concentration levels.

Various aspects of the present invention are described below with reference to the figures. The examples detailed below merely serve to illustrate aspects of the invention and do not in any way limit the scope of the present invention.

DETAILED DESCRIPTION OF ACCOMPANYING FIGURES

Figure 1 illustrates the recombinant ZIKV E antigen in accordance with the present invention. (A) Schematic representation of the ZIKV polyprotein. Ή ₂ N’ and‘COOFF indicate the amino terminus and carboxy-terminus, respectively of the polyprotein. Proteins prM and E are indicated by black and purple boxes, respectively. The region of the polyprotein included in the antigen is bounded by the two white lines in the C- terminal regions of prM and E. (B). Schematic representation of the design of the rec ZIKV E antigen consisting of the last 34 amino acid residues of prM and the first 403 amino acid residues of E. The C-terminally located grey and red boxes denote the pentaglycyl peptide linker and the hexa-histidine (H6) tag, respectively. (C). Predicted amino acid sequence of the recombinant polypeptide is shown in‘B’. The color scheme corresponds to that shown in‘B’. PrMamino acid residues are underlined. The N-terminal dipeptide‘MS’ was introduced during cloning. The downward arrow in‘B’ and‘C’ denotes the signal peptide cleavage site. (D) Map of the rec ZIKV E expression plasmid, pPIC-ZIKV E. The synthetic ZIKV E gene (ZIKV-Envelope) is inserted between the AOX1 promoter (5’ AOX1) on the 5’ side and the transcriptional terminator (TT) on the 3’ side. It carries the selection marker (Zeo ^R ), which is functional in both E. coli and P. pastoris , and the E. coli origin of replication (Ori), for bacterial propagation.

Figure 2 illustrates the comparison of ZIKV and DENV E proteins. Shown is the amino acididentity between the E protein of ZikaSPH2015 (GenBank ID: ALU33341.1) strain, on which the recombinant ZIKV E protein is based, on the one hand, and the E proteins encoded by typical Asian and African ZIKV strains/isolates as well as each typical DENV serotype, on the other. Genbank accession numbers of the polyproteins of each virus is shown in parenthesis below the isolate/strain name. The values in the bottom row represent percent amino acididentity between ZikaSPH2015 E protein and the E proteins of the virus indicated in the cell above. Identity was determined by comparing the amino acid sequences between pairs using NCBI’s protein-protein blastp algorithm.

Figure 3 illustrates the expression screening of transformants. Test-tube cultures of zeocin-positive/f /¾/.s/ /7.stran sfor ants (1-11) were induced with methanol (1.5%) for 3 days and the urea-solubilized M fractions of the total lysates were separated by SDS- PAGE, transferred onto nitrocellulose membrane and probed with mAb 24A12. Aliquots of un-induced P. pastoris cultures were analyzed in lanes‘IT as negative controls. In parallel, two positive controls were also analyzed. One was an expression control represented by a P. pastoris culture of a DENV-1 E-expressing clone (lane‘Di’) and the other was a protein control represented by P. pastoris-ex pressed purified recombinant DENV-3 E protein (lane‘D ₃ ’). Protein ladders were run in lanes‘L\ The sizes (in kDa) of the markers in the protein ladder are indicated on the left of each of the two blots. The arrows on the right denote the position of the recombinant ZIKV E protein. Lane 9 indicates a recombinant ZIKV E-expressing P. pastoris clone.

Figure 4 illustrates the localization and purification of rec ZIKV E protein. (A) Analysis of localization of rec ZIKV E protein in P. pastoris. An aliquot of methanol-induced (Ind) culture of P. pastoris was lysed with glass beads and separated into supernatant ( S) and membrane-enriched pellet ( M) fractions. Total (7) extract prepared from an equivalent aliquot of the induced culture, the ^fraction, and the urea-solubilized M fraction were run on SDS-polyacrylamide gel and subjected to Western blot analysis using mAb 24A12. Total (7) extract prepared from an equivalent aliquot of the un-induced (U) culture was analyzed in parallel. Pre-stained protein ladder was analyzed in lane‘L’. The sizes (in kDa) of the markers in the protein ladder are indicated to the left. The arrow on the right indicates the position of the rec ZIKV E protein. (B) Chromatographic profile of recombinant ZIKV E purification by Ni ²⁺ affinity chromatography, starting from the M fraction of induced cell lysate under denaturing conditions. The solid blue and the dashed black curves represent the profiles of UV absorbance (at 280 nm) and the imidazole step- gradient, respectively. Bound protein was eluted as two peaks (1 & 2). (C) Coomassie- stained SDS-polyacrylamide gel analysis of fractions corresponding to peaks 1 and 2 shown in panel‘B’. Low molecular weight protein ladder was run in lane‘L’. The sizes (in kDa) of the markers in the protein ladder are indicated to the left. The arrow on the right indicates the position of the purified rec ZIKV E protein.

Figure 5 illustrates thephysical characterization of purified rec ZIKV E protein. (A) Coomassie-stained SDS-polyacrylamide gel analysis of pooled peaks 1 and 2 (zE) after dialysis. (B) Western blot analysis of the pooled peaks (zE) using mAb 24A12. In panels A and B, protein ladder was run in lanes‘L’. The sizes (in kDa) of the markers in the protein ladder are shown to the left of each panel. The arrow on the right, of both these panels, indicates the position of the rec ZIKV E protein. (C) The MS N-glycan profile of recombinant ZIKV E protein, obtained using Bruker UltraFlex II MALDI-TOF mass spectrometer. The numbers above/across the peaks indicate the masses of the N-glycan species. The proposed structures of the N-glycan moieties, with the blue squares representing N-acetyl glucosamine and the green circles representing mannose residues, are shown for each of the peaks. (D) Analysis of volume distribution profile of particles in purified rec ZIKV E preparation by DLS. Insets show DLS parameter values obtained based on size distribution by intensity. (E) Transmission electron microscopic image of rec ZIKV E MAPs (the scale is shown on the left lower edge).

Figure 6 illustrates thealum formulation of recombinant ZIKV E MAPs and the immunization schedules. (A) A suspension of recombinant ZIKV E MAPs coated onto alum (20 pg in 100 mΐ) was spun down and 10 mΐ of the resultant supernatant (lane 3) was analyzed by SDS-PAGE. In parallel, aliquots of purified rec ZIKV E MAPs (un-adsorbed on alum) adjusted to 20 pg/100 pi, equivalent to 1 pg (5 pi, lane 2) and 2 pg (10 pi, lane 1) protein, were analyzed for comparison. (B) The alum-formulated rec ZIKV E MAPs were administered by IM injection to BALB/c mice (n= 8) on days 0, 30 and 90 (immunization #1), followed by bleeding on day 105 for nAb titration. The nAb titers in the pooled immune serum against ZIKV (Z), determined by ZIKV -R VP neutralization, and against DENVs 1-4 (Dl, D2, D3 and D4), determined by fluorescence activated cell sorting (FACS)-based virus neutralization assay, are shown in the table below. (C) The alum-formulated recombinant ZIKV E MAPs were administered by IP injection to BALB/c mice («=14) on days 0, 14 and 28 (immunization #2), followed by bleeding on day 38 for nAb titration. To assess memory recall response, a subset of these mice («=5) was rested for ~4 months and bled (on day 159), followed by a 4 ^th dose of immunization (day 160) and bleeding 10 days later (day 170). ZIKV-specific nAb titers in pooled sera from days 38, 159 and 170 are shown in the table below. In both panels‘B’ and‘C’, mock-immunizations were performed in parallel using PBS instead of rec ZIKV E MAP during alum formulation (Ctrl), and sera from these included in nAb titrations as shown in the tables.

Figure 7 illustrates theimmunological evaluation of rec ZIKV E MAP-induced antibodies. (A) Pooled serum collected from BALB/c mice («=8) immunized (IM) on days 0, 30 and 90, with alum-formulated rec ZIKV E MAPs was analyzed by indirect ELISA using purified P. pastoris- produced rec E proteins corresponding to ZIKV (purple), DENV-1 (magenta), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) as coating antigens. (B) Pooled serum from BALB/c mice («=14) immunized (IP) on days 0, 14 and 28, with alum-formulated rec ZIKV E MAPs was analyzed by indirect ELISA using the five rec E proteins listed in panel‘A’. (C). Pooled serum described in panel‘A’ was analyzed in indirect ELISAs using purified E. co/z-produced MBP-EDIII fusion proteins corresponding to ZIKV (purple), DENV-1 (magenta), DENV-2 (green), DENV-3 (blue) and DENV-4 (black) as coating antigens. (D) Pooled serum described in panel‘B’ was analyzed in indirect ELISAs using purified E. co/z-produced MBP-EDIII fusion proteins listed in panel‘C’. One of two independent experiments is shown. Each data point represents the average of duplicates.

Figure 8 illustrates theevaluation of DENV enhancement by BALB/c anti-rec ZIKV E MAP antiserum in vitro. (A & B). DENV-1 (magenta), DENV-2 (green), DENV-3 (blue) or DENV-4 (black) were pre-incubated with serial dilutions of pooled sera obtained either from mock-immunized (PBS+alum) mice (panel‘A’) or from mice immunized with alum-formulated rec ZIKV E MAPs (panel ‘B’) and used for infecting K562 cells (immune sera were from mice immunized on days 0, 14 and 28). Percent DENV-infected cells were determined by flow cytometry. (C) Experiment similar to those described in panels‘A’ and‘B’, except that DENVs were pre-incubated with serial dilutions of mAb 4G2 instead of murine immune serum before K562 infection.

Figure 9 illustrates theevaluation of the enhancement potential of anti-rec ZIKV E MAP antibodies in vivo. (A) Schematic representation of the ADE model based on the AG129 mouse. IC generated in vitro by pre-incubating a sub-lethal dose of DENV-2 S221 with immune serum is inoculated into AG129 mice (n=6) followed by survival monitoring for 12 days. (B) Kaplan-Meier survival curves for groups (z/=6) of AG129 mice which were intravenously administrated with a sub-lethal dose of DENV-2 S221 (pre-incubated with NMS, green curve) or with ICs generated in vitro by mixing the sub-lethal dose of DENV-2 S221 either with mAb 4G2 (grey curve), anti -DENV-2 antiserum (black, dashed curve), or serum from BALB/c immunized (on days 0, 14, 28) with rec ZIKV E MAPs (purple curve). Survival was observed for 12 days post-challenge. Survival of mice in the DENV-2 S221+anti-rec ZIKV E MAP antiserum group (purple curve) and DENV-2 S221+NMS group (green curve) were comparable to each other (p=0.5282) and significantly higher (/ =().0095) than that in the DENV-2 S221+4G2 group (grey curve) or DENV-2 S221+anti -DENV-2 antiserum group (black, dashed curve), based on the Log- Rank (Mantel-Cox) test.

Figure 10 illustrates them vivo ADE model utilized to assess the ZIKV-enhancement potential of anti-rec ZIKV E MAP antibodies. (A) Immune sera were injected into riVa/ mice (derived from C57BL/6 strain) two hours before ZIKV challenge. Mice were monitored over 12 days post sub-lethal ZIKV infection for survival, clinical symptoms and weigh loss. (B-D) Stall ¹ mice were inoculated IP with NMS (dotted curve), anti- DENV-3 immune serum (dashed curve, black) or anti-rec ZIKV E MAP antiserum (solid curve) and then challenged with ZIKV (PRVABC59) two hours later. Mice were monitored for survival (panel Έ’), clinical symptoms (panel‘C’) and weight loss (panel ‘D’) for a period of 12 days following ZIKV infection. Survival of mice in the NMS and anti-rec ZIKV MAP antiserum groups (panel‘B’) was not statistically different from each other (p= 0.2636).

The invention is now illustrated by way of non-limiting examples.

Example 1: Design and expression of rec ZIKV E protein

The synthetic ZIKV E gene, which was codon-optimized for expression in P. pastoris , encoded the last 34 amino acid ( aa ) residues of the ZIKV prM protein, to serve as the signal peptide, followed by the first 403 aa residues of the full-length ZIKV E protein, the vaccine antigen. Nucleotide sequences encoding a penta-glycine peptide linker followed by a hexa-histidine tag were appended at the 3’ end of the ZIKV E gene. This synthetic gene which was placed under the P. /¾/.s/o/7.sm ethanol -i nducibl e alcohol oxidase 1 ( AOX1 ) promoter of the pPICZ A vector (Figure l)is predicted to encode a protein of ~49 kDa in its unprocessed form, which upon signal peptide cleavage exists as a ~45 kDa protein (Table 1). Table 1 : Properties of recombinant ZIKV E protein

The ZIKV E ectodomain-encoding sequences of the synthetic ZIKV E gene were derived from the ZikaSPH2015 Brazilian strain. ZikaSPH2015 E protein displays at least 99% sequence identity with E proteins of Asian ZIKV strains, at least 96% sequence identity with E proteins of African ZIKV strains and 55-57% sequence identity with respect to E proteins of the four DENVs (Figure 2).

Plasmid pPIC-ZIKV E (pPICZ A vector into which the synthetic ZIKV E gene was inserted in the polylinker) was integrated into the genome of P. pastoris strain KM71H by electroporation according to the vendor’s manual (Invitrogen Life Technologies, Thermo Fisher Scientific). The resultant transformants, obtained through zeocin selection, were subjected to an expression screening step to identify clones capable of expressing the rec ZIKV E protein. Rec ZIKV E protein was identified with an‘ in-house’flavivirus E- specific mAb 24A12 in Western blots (Figure 3). This Western blot may also be probed with the flavivirus-specificmAb 4G2 (Henchal EA et al, Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg. 1982; 3: 830-836) or any of the ZIKV E- specific mAbs described in the literature (Zhao H et al, Structural basis of Zika virus- specific antibody protection. Cell. 2016; 166: 1016-1027;Stett\er et al, Specificity, cross reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826; Wang Q et al, Molecular determinants of human neutralizing antibodies isolated from a patient infected with Zika virus. SciTransl Med. 2016; 8: 369ral79 ) or ZIKV- infected polyclonal animal or patient sera, as must be apparent to one skilled in the art. The localization of the induced rec ZIKV E protein was examined by analyzing the supernatant (Sup) and denaturant-solubilized membrane-enriched pellet (M) fractions of the induced lysate using the same Western blot assay (Figure 4A). This revealed that rec ZIKV E is exclusively present in the insoluble fraction of the total lysate.

Example 2: Purification of rec ZIKV E protein

Rec ZIKV E protein was purified using Ni ²⁺ -NTA affinity chromatography, taking advantage of the C-terminally engineered 6x-His tag. As it was associated with the insoluble M fraction of induced lysates, purification was performed under denaturing conditions. 6M guanidine hydrochloride (Gu-HCl) was preferred for extraction compared to 8M urea as the former was twice as efficient as the latter. However, as Gu-HCl- containing column fractions are not compatible with SDS-PAGE analysis, the denaturant was switched from Gu-HCl to urea after column binding as described below.

A 2 L culture of P. pastoris harboring the ZIKV E gene grown to logarithmic phase was induced with 1.5 % methanol for 3 days at 30°C. This culture was centrifuged and the resultant induced cell pellet (~50 g wet weight) was washed in sterile l x phosphate buffered saline (PBS) and re-suspended in 300 ml cell suspension buffer, CSB (50mM Tris-HCl (pH 8.5)/500mM NaCl/1 mM phenyl methyl sulfonyl fluoride). This suspension was subjected to lysis in a Dynomill (WAB, Muttenz, Switzerland) and clarified by centrifugation at 16,000 xg to separate out the membrane-enriched M fraction. The M fraction was stirred in 200 ml membrane extraction buffer MEB-1 [CSB supplemented with 6M guanidine hydrochloride (Gu-HCl) and 20 mM imidazole] for ~4 hours at room temperature (RT). The resultant extract was centrifuged (13,000 rpm, 1 hour, RT) and filtered (0.45 m). The resultant clarified extract was bound to Ni ²⁺ -NTA resin (25 ml of a 50% slurry, pre-equilibrated in MEB-1) in batch mode (at RT overnight). The Ni ²⁺ -NTA resin plus bound sample slurry was packed into a chromatographic column which was connected to an AKTA purifier. The column was washed extensively with MEB-1 followed by MEB-2 (CSB supplemented with 8M urea and 20 mM imidazole) to replace 6M Gu-HCl with 8M urea. Elution was performed using a step imidazole gradient in MEB-2. Column fractions were analyzed by SDS-PAGE.

Elution resulted in the emergence of two peaks, one at -100 mM and the other at -250 mM imidazole (Figure 4B). SDS-PAGE analysis revealed the presence of a ~45kDa protein in all fractions across both peaks 1 and 2 (Figure 4C). This essentially reflects that 100 mM imidazole was adequate to displace only a small fraction of the bound recombinant protein, with more effective elution occurring at 250 mM imidazole. Thus both peaks were pooled together, dialyzed against 20 mMTris-HCl (pH 8.5) buffer containing 50 mMNaClto remove urea and imidazole and stored frozen in aliquots until use. Starting from a 2L shake-flask culture (-50 g induced biomass), the typical yield of purified recombinant protein was -50 mg (~lmg purified protein/gram wet weight of induced cell biomass).Purity was assessed to be >95% based on image analysis (Bio Rad Gel Doc EZ imager). It is well established that the P. pastoris expression system is associated with very high recombinant protein yields (Cereghino&Cregg, Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. 2000; 24: 45-66; Ahmad M et al, Protein expression in Pichia pastoris : recent achievements and perspectives for heterologous protein production. ApplMicrobiolBiotechnol. 2014; 98: 5301-5317; Gurramkonda C et al, Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris : application to intracellular production of hepatitis B surface antigen. Microb. Cell Fact. 2009; 8: 13). High cell density cultivation under carefully controlled conditions can yield recombinant proteins in gram quantities per liter of culture. If one were to assume a single dose of vaccine to contain 20 pg recombinant vaccine antigen, 1 g of recombinant protein made using P. pastoris would translate into 50,000 vaccine doses.

Example 3: Physical characterization of purified rec ZIKV E protein

An aliquot of the pooled Ni ²⁺ -NTA affinity-purified material was analyzed by SDS- PAGE and found to contain a single protein band of -45 kDa (Figure 5 A). This band was recognized by mAb 24A12, which recognizes ZIKV EDIII, in a Western blot (Figure 5B). mAb 24A12 in this step may be replaced with others antibodies including, but not limited to, flavivirus-specific mAb 4G2, ZIKV specific mAbs ZV-48, ZV- 64, ZV-67, polyclonal animal sera or ZIKV-infected human sera. As mentioned above a prM signal peptide (34 ad) was included at the N-terminus of the recombinant protein to ensure proper processing. N-terminal sequence analysis revealed that the prM signal peptide had been cleaved off from the P. pastoris-ex pressed rec ZIKV E protein. N-linked glycan analysis of the purified rec ZIKV E protein was carried out using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MS glycan profile, as well as the mass composition and relative abundance of the N-glycan moieties of rec ZIKV E protein are summarized in Figure 5C. Oligomannose structures up to Manl3 were observed with Man9 being the most abundant species.

Reports in the literature show that it is necessary to co-express ZIKV E together with ZIKV prM to obtain virus-like particles (VLPs). Such ZIKV VLPs have been observed using insect cells (Dai S et al, Zika virus baculovirus-expressed virus-like particles induce neutralizing antibodies in mic e.VirolSinica 2018; 33: 213-226 ) and mammalian cells (Boigard H et al, Zika virus-like particle (VLP) based vaccin e.PLoSNegl Trop Dis. 2017; 11: e0005608; Garg H et al, Development of virus-like-particle vaccine and reporter assay for Zika virus. J Virol. 2017; 91: e00834-17; Salvo MA et al, Zika virus like particles elicit protective antibodies in mice. PLoSNegl Trop Dis. 2018; 12: e0006210; Urakami A et al, An envelope-modified tetravalent dengue virus-like particle vaccine has implications for flavivirus vaccine design. J Virol. 2017; 91: eO 1181-17) as expression hosts. On the other hand, expression of ZIKV E alone has been tested using E. coli (Han JF et al, Immunization with truncated envelope protein of Zika virus induces protective immune response in mice. Sci Rep. 2017; 7: /dd-/ 7; Liang H et al, Recombinant Zika virus envelope protein elicited protective immunity against Zika virus in immunocompetent mice. PLoS One 2018; 13: e0194860) and insect cells (To et al, Recombinant Zika virus subunits are immunogenic and efficacious in mice. mSphere 2018; 3: e00576-l 7; Liang H et al, Recombinant Zika virus envelope protein elicited protective immunity against Zika virus in immunocompetent mice. PLoS One 2018; 13: eO 194860) as host systems. While E. coli expression led to deposition of the recombinant ZIKV E into inclusion bodies, insect cell expression resulted in soluble forms of ZIKV E. Interestingly, none of these reported the formation of VLPs. To examine if ZIKV E expressed in P. pastoris in the absence of ZIKV prMmay possess the ability to form higher order structures through self-assembly, the following studies were undertaken. Laser-based dynamic light scattering (DLS) analysis was performed using Zetasizer Nano ZS90 (Malvern instruments, Malvern, UK) after adjusting the concentration of purified rec ZIKV E protein to 300 pg/ml in 20 mMTris-HCl/50 mMNaCl (pH 8.5). DLS analysis revealed a single major homogenous particle peak (Pdl=0.359), suggestingthat the purified ZIKV E preparation does indeed self-assemble into discrete higher order structures (Figure 5D). This was corroborated by electron microscopic (EM) examination of the purified rec ZIKV E protein preparation. For this the purified preparation was adjusted to a protein concentration of 5-10 pg/ml (in 20 mMTris-HCl/50 mMNaCl, pH 8.5), applied onto carbon-coated EM grid (1-2 min), negatively stained with 1% uranyl acetate (30 sec) and washed (using Milli-Q water) before visualization under a Tecnai EM. The EM visualization showed discrete particles of 30-50 nm (Figure 5E). Collectively, these data demonstrate that purified rec ZIKV E protein prepared using the P. pastoris host system self-assembles into particulate higher order structures in the absence of any ZIKV prM protein. Self-assembly of the rec ZIKV E protein into discrete higher order structures is unexpected and novel, given that this has not been observed before (Han JF et al , Immunization with truncated envelope protein of Zika virus induces protective immune response in mice. Sci Rep. 2017; 7: 10047; Liang H et al, Recombinant Zika virus envelope protein elicited protective immunity against Zika virus in immunocompetent mice. PLoS One 2018; 13: e0194860; To A et al, Recombinant Zika virus subunits are immunogenic and efficacious in mice. mSphere 2018; 3: e00576-17). Further, it is also unexpected that discrete higher order particulate structures were observed in the absence of ZIKV prM, in contrast to the reported observation which suggest the requirement of ZIKV prM for ZIKV E to form higher order structures (Boigard H et al, Zika virus-like particle (VLP) based vaccine. PLoSNegl Trop Dis. 2017; 11: e0005608; Garg H et al, Development of virus-like-particle vaccine and reporter assay for Zika virus. J Virol. 2017; 91: e00834-17; Salvo et al, Zika virus like particles elicit protective antibodies in mice. PLoSNegl Trop Dis. 2018; 12: e0006210; Urakami A et al, An envelope-modified tetravalent dengue virus-like particle vaccine has implications for flavivirus vaccine design. J Virol. 2017; 91: eO 1181-17; Dai L et al, Zika virus baculovirus-expressed virus-like particles induce neutralizing antibodies in mic e. VirolSinica 2018; 33: 213-226). These higher order structures differ in certain key aspects from the ZIKV prM + E-containing VLPs described in literature (Boigard H et al, Zika virus-like particle (VLP) based vaccine. PLoSNegl Trop Dis. 2017; 11: e0005608; Garg H et al, Development of virus-like-particle vaccine and reporter assay for Zika virus. J Virol. 2017; 91: e00834-17; Salvo MA et al, Zika virus like particles elicit protective antibodies in mice. PLoSNegl Trop Dis. 2018; 12: e0006210; Urakami et al, An envelope-modified tetravalent dengue virus-like particle vaccine has implications for flavivirus vaccine design. J Virol. 2017; 91: eOl 181-11). These higher order structures (i) contain rec ZIKV E retrieved from the membrane-enriched fraction of the P. pastoris lysate, which includes only the ZIKV E ectodomain (not the full-length ZIKV E protein) and (ii) lack prM and may consequently manifest subtle differences in the relative disposition of the rec ZIKV E molecules (and its domains) within the higher order structure. Consequently, the P. pastoris-ex pressed rec ZIKV E-containing higher order structures are defined as membrane-associated particles (MAPs).

Example 4: Antigenic characterization of ZIKV MAPs

As the relative disposition of rec ZIKV E in the MAPs described above may be different from that of full-length ZIKV E in the conventional ZIKV prM + E-containing VLPs reported in literature, it is essential to probe the rec ZIKV E antigenic determinants accessible on the MAPs, to gauge its suitability as an effective immunogen. Thus, these ZIKV MAPs were probed with a panel of ZIKV-specific human and murine conformational mAbs. Since potent ZIKV-specific neutralizing epitopes map to EDIII, a set of ZIKV EDIII-specific mAbswere deployed to probe the ZIKV MAPs using an indirect ELISAs approach as described below.

ELISA microtiter wells were coated with purified rec ZIKV E MAPs in 0.1 M sodium bicarbonate buffer (pH 9.6), overnight at 4°C (0.2 pg/0.1 ml/well). In parallel, wells were also coated with P. pastoris produced purified rec DENV-2 E protein, for comparison. Coated wells were washed three times with lx PBS containing 0.1% Tween-20 (PBST) and blocked with 5% skim milk (200pl/well), prepared in lx PBS, for 2 hours at 37°C. Wells were washed again with PBST (3x) and treated with the indicated ZIKV EDIII- specific mAbs (0.1 pg/O.l ml/well, prepared in 2.5% skim milk) for 1 hour at 37°C. Again, wells were washed with PBST (6x) and incubated with anti-mouse IgG horseradish peroxidase (HRPO) conjugate (0.1 mg/0.1 ml/well, prepared in 2.5% skim milk) for 1 hour at 37°C. Once again, wells were washed with PBST (6x) and treated with 3, 3’, 5, 5’ tetramethylenebenzidine (TMB) substrate (0.1 ml/well) for 30 minutes at RT. The reaction was stopped with 1 N H ₂ SO ₄ (0.1 ml/well) and the absorbance read at 450 nm, with 650 nm as a reference. The data are summarized in Table 2.

*values shown are absorbances at 450nm

The rec ZIKV E protein-containing MAPs were recognized efficiently by the murine mAbs ZV-48 and ZV-67, but not by ZV-2. All three are ZIKV-specific murine mAbs. None of them recognized recombinant DENV-2 E protein. The binding sites of these mAbson the ZIKV E protein have been mapped using recombinant protein-binding studies in conjunction with X-ray crystallography (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027). ZV-2, ZV-48 and ZV-67 bind to the ABDE sheet, the C-C’ loop, and the lateral ridge (LR), respectively of ZIKV EDIII. Of these, the LR epitope is quite complex in that it is comprised of several secondary structure elements that include the A-strand, the B-C loop, D-E loop and F-G loop involving 21 contact residues (Zhao H et al, Structural basis of Zika virus-specific antibody protection. Cell. 2016; 166: 1016-1027). The efficient recognition by mAb ZV- 67, a potent neutralizer of ZIKV infectivity, suggests that the antigenic integrity of the LR epitope is largely preserved in the rec ZIKV E MAPs. The binding of mAh ZV-48 (also a strong neutralizer of ZIKV infectivity, but not as potent as ZV-67), which recognizes another distinct secondary structural element, the C-C’ loop, suggest subtle difference(s) between rec ZIKV E-containing MAP and the ZIKV particle, as the C-C’ loop is not predicted to be accessible on the virion surface (KostyuchenkoVAe/ al, Structure of the thermally stable Zika virus. Nature. 2016; 533: 425-428; SirohiDe/ al, The 3.8 A resolution cryo-EM structure of Zika virus. Science. 2016; 352: 467-470). On the other hand, ABDE sheet recognized by mAb ZV-2, a weak neutralizer, and also not predicted to be accessible on the ZIKV particle (Kostyuchenko VA et al, Structure of the thermally stable Zika virus. Nature. 2016; 533: 425-428; SirohiDe/ al, The 3.8 A resolution cryo- EM structure of Zika virus. Science. 2016; 352: 467-470 ), was not detectable on the rec ZIKV E MAPs as well. The rec ZIKV E MAPs were also recognized by a human mAb, ZKA-64 (highly potent nAb), specific to ZIKV EDIII (StettlerKe/ al, Specificity, cross reactivity, and function of antibodies elicited by Zika virus infection. Science. 2016; 353: 823-826). Taken collectively, the mAb probing data suggest that the rec ZIKV EMAPs preserve the overall antigenic integrity of EDIII, but with subtle differences.

Further, to those skilled in the art, it will be evident that the data in Table 2 also attest to the utility of the rec ZIKV MAPs as antigens for the specific detection of antibodies against ZIKV that may be present in body fluids and tissues as a consequence of ZIKV infection.

Example 5: Investigation of the capacity of P. yastoris- produced rec ZIKV E MAPs to induce ZIKV-specific, ZIKV EDIII-directed antibody response

That the P. pastoris-e pressed rec ZIKV E forms MAPs displaying ZIKV EDIII (particularly the LR epitope) attests to the vaccine potential of these MAPs. To investigate this, BALB/c mice were immunized with the rec ZIKV E-containing MAPs formulated with alum as adjuvant. Groups of 4-6 week old BALB/c mice were either mock-immunized (500 pg alhydrogel in 100 mΐ lx PBS), or immunized with alhydrogel- formulated rec ZIKV E MAPs. Two immunization schedules, with 3 doses in either schedule, administered over 3 months in one, and over 1 month, in the other were adopted (Figure 6). In both these immunization schedules, a single immunization dose consisted of 20 pg purified rec ZIKV E MAPs adsorbed onto 500 pg alhydrogel in a volume of 100 mΐ lx PBS.

Sera were collected 10-15 days after the 3 ^rd dose for determination of total Ab titers by indirect ELISA, using 5 different recombinant flaviviralE proteins (corresponding to ZIKV and DENV serotypes 1-4) as the capture antigens. For coating, ELISA microtiter wells were treated overnight at 4°C with purified recombinant protein in 0.1 M sodium bicarbonate buffer, pH 9.6 (0.2 pg/0.1 ml/well). Coated wells were washed with lx PBS containing 0.1% Tween-20 (PBST) and blocked for 2 hours at 37°C, with 5% skim milk prepared in lx PBS. The blocked wells were washed (3x with PBST) and treated (0.1 ml/well, 1 hour, 37°C) with serial 2-fold dilutions of different immune sera. Wells were washed (6x with PBST) and incubated with 0.1 ml anti -mouse IgG HRPO conjugate (0.1 pg/ml in 2.5% skim milk, prepared in IX PBS) for 1 hour at 37°C. Again, wells were washed (6x with PBST), treated with TMB substrate (0.1 ml/well) for 30 minutes at 37°C, followed by 1 N H ₂ S0 (0.1 ml/well). The absorbance was read at 450 nm, with 650 nm as reference. Background ELISA absorbance values were < 0.05. ELISA end-point titers, defined as the highest reciprocal serum dilution that yielded an absorbance 4-fold over background (that is, a cut-off absorbance value of 0.2), were determined from extrapolation of non-linear regression curves using GraphPad Prism (v7.0) software.

The indirect ELISA data for total Ab titers in the immune sera are depicted in Figure 7. Interestingly, both immunization schedules resulted in the elicitation of high Ab titers (serum logio titers were >10 ⁵ ) specific torec ZIKV E (Figure 7, panels A and B). Consistent with the antigenic similarity which exists between ZIKV E and the E proteins of the four DENV serotypes (Figure 2), the anti-rec ZIKV E MAP antisera manifested cross-reactivity against the DENV E proteins. Once again this cross -reactivity was observed in both immunization schedules. In both instances, the DENV E-cross-reactive end-point titers were more than an order of magnitude lower than those against rec ZIKV E MAPs. Table 2 data revealed that the rec ZIKV E MAPs display EDIII, as evidenced by its efficient recognition by ZIKV ED Ill-specific mAbs. Further experiments were conducted to determine what fraction of the total Ab titers elicited by rec ZIKV E MAPs are specific to ZIKV EDIII. Another series of indirect ELISAs were carried out wherein the microtiter wells were coated with recombinant ZIKV EDIII fusion protein, instead of rec ZIKV E protein, as coating antigen. In fact, five different MBP -EDIII fusion proteins were used. The domain III moieties, of these five MBP-EDIII fusion proteins, corresponded to the five flaviviral E proteins used above, that is E proteins of ZIKV, DENV-1, DENV-2, DENV-3 and DENV-4. This was done to assess the relative proportions of ZIKV and DENV EDIII-specific Ab titers in the BALB/c immune sera. The data are shown in Figure 7, panels C and D. This revealed that the Ab titers against recombinant ZIKV EDIII protein were quite high (serum logio titers were >5) and very similar to titers against rec ZIKV E protein. Again, both immunization schedules yielded similar results. Importantly, these data provided two important and critical pieces of information: (i) practically all of the Ab titers elicited by rec ZIKV E MAPs are directed to ZIKV EDIII; and (ii) cross-reactivity of the rec ZIKV E MAP -induced Abs towards DENV ED Ills was extremely low. Two significant conclusions arising from these data are that: (i) rec ZIKV E MAPs elicit predominantly ZIKV EDIII-focused Ab responses, indicating that these MAPs display ZIKV EDIII on the surface; and (ii) the ZIKV EDIII-specific Abs elicited by rec ZIKV E MAPs are essentially ZIKV-specific.

Example 6: Investigation of the capacity of P. yastoris- produced rec ZIKV E MAP- induced Abs to neutralize ZIKV

The capacity of the ZIKV EDIII-directed antibodies, elicited by rec ZIKV E MAPs, toblock the infectivity of ZIKV was assessed. For this, ZIKV reporter virus particles (RVPs) were used instead of wild-type ZIKV. These ZIKV RVPs are identical to wild- type ZIKV on the outside and infect susceptible cells once but do not produce infectious progeny as they lack the structural genes. Instead they encode Renilla luciferase reporter whose activity provides a read-out of ZIKV RVP entry into cells (Shan C et al, Evaluation of a novel reporter virus neutralization test for serological diagnosis of zika and dengue virus infection. J ClinMicrobiol. 2017; 55: 3028-3036; Shan C et al, A rapid Zika diagnostic assay to measure neutralizing antibodies in patients. EBioMed. 2017; 17: 157-162). These ZIKV RVPs were pre-incubated with serial dilutions of the anti-rec ZIKV E MAP antiserum, obtained from the two different immunization schedules, followed by determination of residual infectivity in terms of luciferase activity.

The ZIKV RVP inhibition assay was performed as follows. Vero cells were seeded in 96- well plates at 1.5xl0 ⁴ /well/200 mΐ a day in advance (day 0). Replicates of ZIKV RVPs (10 mΐ each) were pre-incubated with serial two-fold dilutions of heat-inactivated (D, 56°C, 30 min) immune sera, in a total volume of 100 mΐ, for 1 hour. On day 1, Vero cells were exposed to this pre-incubation mixture (100 mΐ ZIKV-RVP + immune serum/well). After allowing 1 hour of adsorption time at 37°C, the cells were fed with D-MEM+2% AFBS (150 mΐ/well), and allowed to incubate at 37°C in a humidified 10% C0 ₂ incubator. Control wells were set up in parallel in which the antiserum was omitted in the pre incubation step. On day 3 post-exposure to ZIKV-RVP, luciferase activity was measured as follows. Media was aspirated off from the wells which were rinsed once with lx PBS and followed by the addition of freshly diluted (in lx PBS containing 10% AFBS, final concentration 60 mM) ViviRen Live Cell substrate (100 mΐ/well). Luminiscence was measured 2 minutes after substrate addition using SpecrtaMax M3 microplate reader (Molecular Devices, USA) set to‘luminiscence’ read mode. ZIKV neutralizing activity was determined in terms of RNT ₅₀ titer, defined as the serum dilution capable of causing a 50% reduction in the ZIKV RVP-expressed luciferase activity with reference to the activity expressed by ZIKV-RVP, in the absence of immune serum, taken asl00%

Data from this experiment are depicted in the tabular format in Figure 6 (tables below panels B and C). Interestingly, this experiment revealed that the antibodies elicited by rec ZIKV E MAPs neutralized the ZIKV RVPs effectively. Also, the rapid immunization schedule did not adversely affect the ZIKV nAb titer. In parallel, the nAb titers against each of the four DENV serotypes were also determined using a FACS-based assay (Kraus AA et al, Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization. J ClinMicrobiol. 2007; 45: 3777-3780). This experiment revealed that the Abs elicited by rec ZIKV E MAPs, which neutralized the ZIKV RVPs effectively, did not have any neutralizing potency against any of the four DENVs (Figure 6B). This is a surprising finding given the similarity of DENVs to ZIKV. Apparently, the rec ZIKV E MAPs elicit solely ZIKV-specific nAbs.

Further experimentation was conducted to determine whether the ZIKV-specific nAb response could be re-called. A subset of mice («=5) in the shorter immunization experiment, were allowed to rest for >4 months after the 3 ^rd immunization, and then given a 4 ^th dose (day 160). Mice were bled one day before (day 159) and 10 days after (day 170) the 4 ^th dose for nAb estimation using the ZIKV RVP luciferase assay (Figure 6C). This experiment demonstrated >4-fold increase in resting titers following recall (RNT ₅₀ = 55 before and 241 after recall). This reflects a specific recall response, and is indicative of the generation of memory B cells as a result of the primary immunization.

The predominant Ab response to rec ZIKV E MAPs is directed to EDIII (indirect ELISA data, Figure 7). To determine the contribution of ZIKV EDIII- specific Abs to the observed nAb titers, EDIII-specific Abs were specifically depleted from the anti-rec ZIKV E MAP antiserum on immobilized ZIKV EDIII as follows. Amylose resin was washed 3x with sterile water and 3x with column buffer (20 mMTris-HCl/200 mMNaCl/1 mM EDTA, pH 7.4). For each washing step, the resin was spun at 3,000 rpm for 5 min at RT. To immobilize MBP and MBP-fused to ZIKV EDIII, these proteins were separately incubated with the washed resin (300 pg protein/100 mΐ resin/900 mΐ column buffer) overnight at 4°C on a rocking platform. Next day, the resin was washed 3x with column buffer and 3x with lx PBS. The protein bound resin was blocked with 1% bovine serum albumin (BSA) prepared in lx PBS for 2 hours at RT. Blocked resin was again washed 3x with lx PBS. Anti rec-ZIKV E MAP immune sera (1 : 10 diluted in lx PBS, heat inactivated) was separately incubated with MBP and MBP-ZIKV EDIII fusion protein- bound amylose resin (400 mΐ sera/100 mΐ resin + bound protein) for 45 minutes at 37°C on a rocking platform. Following this, the samples were spun down (3, 000 rpm / 5 minutes/RT) and used for indirect ELISA (using MBP-ZIKV EDIII fusion protein as the coating antigen) and RNT ₅₀ determination using ZIKV RVP assay. ZIKV EDIII-specific Ab depletion experiment was performed as described above, using the day 38 and day 170 immune sera from the memory recall experiment (Figure 6C). Indirect ELISA revealed that the total ZIKV EDIII-specific Ab titers of both sera, which were not significantly affected following mock depletion using immobilized MBP, underwent considerable decrease following depletion on immobilized MBP ZIKV EDIII fusion protein. For example at a 1000-fold dilution of serum, ELISA absorbance values decreased >50% following depletion on immobilized MBP ZIKV EDIII fusion protein. As a next step, nAb titers in the un-depleted and MBP ZIKV EDIII-depleted immune sera were determined. After one round of depletion, the nAb titers of the day 38 (RNT ₅₀ , before=251; RNT ₅₀ , after=147) and day 170 (RNT ₅₀ , before=301; RNT ₅₀ , after=94) immune sera decreased by -48% and -70% upon depletion of ZIKV EDIII-specific Abs.ZIKV-specific nAb titers can be abrogated to as high as 80% by additional rounds of depletion of the immune sera on immobilized ZIKV EDIII. It is evident that depletion of ZIKV EDIII-specific Abs in the immune sera strongly correlates with a decrease in nAb titer. These data suggest a role for ZIKV EDIII-specific Abs in ZIKV neutralization and also show that these Abs comprise a significant proportion of the memory recall response.

Example 7: Evaluation of flavivirus infection-enhancing capacity of rec ZIKV E MAP-induced Abs

The serological cross-reactivity among flaviviruses, stemming from the close phylogenetic proximity, poses the risk of ADE. Cross-reactive Abs to one flavivirus can potentially enhance infection by another flavivirus by binding to the latter and facilitating its uptake v/aFcyR-bearing cells. In the context of the existence of the phenomenon of ADE in flaviviral infections, it is important to evaluate if the rec ZIKV E MAP -induced DENV E-cross-reactive Abs observed (Figure 7, panels A and B), seen to lack any DENV-neutralizing activity, possess infection-enhancing potential. To this end, the ADE potential of anti-rec ZIKV E MAP antiserum using both in vitro and in vivo assays was evaluated.

In vitro DENV ADE assays were performed using the FcyllR-expressing K562 cell line. DENV-1, -2, -3 & -4 (40 mΐ/well, moi=l) were incubated separately with serial two-fold dilution of heat inactivated anti-rec ZIKV E MAP antiserum, mock (PBS)-immune serum, and 4G2mAb (40 mΐ/well) for 1 hour at 37°C. Virus and serum dilutions were prepared in D-MEM+2% AFBS. K562 cells (5X10 ⁴ cells/20 mΐ/well) were mixed with the immune complex (IC) and allowed to incubate for 1 hour at 37°C in a humidified 10% CO2 incubator (final volume of 100 mΐ/well). Next, the cells were washed once with D- MEM+2% AFBS and suspended in fresh D-MEM+2% AFBS (200 mΐ/well) and incubated for 24 hours at 37°C in a humidified 10% C0 ₂ incubator. Next day, cells were fixed with 4% formaldehyde (50 mΐ/well, 10 minutes, RT), permeabilized (with BD Perm/Wash), blocked with normal mouse serum (NMS) (40 mΐ/well, 20 min, RT) and stained with prM-specific 2H2-Alexa 488 conjugate (20 mΐ/well, 1 hour, 37°C). Cells were analyzed in BD FACS-Verse (BD Biosciences). The results are presented in Figure 8.

In the in vitro experiment, the capacity of serially diluted sera from mock-immunized mice and alum-formulated rec ZIKV E MAP -immunized mice to enhance the infection of K562 cells by each DENV serotype was analyzed. This experiment revealed that none of the DENVs could infect K562 cells over a wide range of anti-rec ZIKV E MAP antiseradilution (10 ¹ to 10 ⁵ ). This is a striking finding, given that rec-ZIKV E MAP- induced Abs did bind to recombinant E proteins of the four DENV serotypes (Figure 7, panels A & B). As expected, a control experiment carried out in the presence of varying concentrations of mAh 4G2, in place of the immune serum, showed ADE with each of the four DENVs, although to different extents. This mAh, specific to the highly conserved flaviviral FLE, is documented to manifest ADE activity (Watanabe S et al, Dengue virus infection with highly neutralizing levels of cross-reactive antibodies causes acute lethal small intestinal pathology without a high level of viremia in mice. J Virol. 2015; 89: 5847-5861). Collectively, the data show that rec-ZIKV E MAP-induced Abs, which potently neutralize ZIKV specifically, neither neutralize nor enhance DENV infections in vitro.

Further experiments were conducted to evaluate therelevance of the data on ADE obtained with K562 cells to the in vivo situation.For this purpose, the double interferon a/b and g receptor knock-out AG129 mouse-based ADE model which is susceptible to DENV infectionwas employed (Figure 9A). This mouse can sustain a sub-lethal DENV-2 infection without succumbing to it. But in the presence of an enhancing Ab, the sub-lethal DENV-2 infection escalates to a lethal infection (Watanabe S el al, Dengue virus infection with highly neutralizing levels of cross-reactive antibodies causes acute lethal small intestinal pathology without a high level of viremia in mice. J Virol. 2015; 89: 5847-5861). To assay in vivo ADE of DENV-2 using the AG129 mouse model, ICs were first generated in vitro by pre-incubating (1 hour on ice) a sub-lethal dose of DENV-2 S221 (2xl0 ⁴ FACS Infectious Units) separately with 5 mΐ (neat) of either NMS or anti-rec ZIKV E MAP-antiserum in a total volume of 50 mΐ. Control IC was generated in parallel by using either 10 pg mAh 4G2, or 5 mΐ of anti -DENV-2 antiserum, during pre-incubation with DENV-2 S221. All the different ICs were separately inoculated, intravenously (retro-orbitally), into groups (n= 6) of 6-8 week old AG129 mice. The mice were monitored twice daily for mortality for up to 12 days. When these mice were injected with IC generated by pre-incubating a sub-lethal dose of the challenge strain DENV-2 S221 with mAh 4G2 (DENV-2 S221+mAb 4G2 IC) or anti-DENV-2 antiserum (DENV-2 S221+anti-DENV-2 antibody IC), there was 100% mortality by days 5-6 post-challenge, in contrast to mice in the virus control (VC) group that received the same sub-lethal dose of DENV-2 S221, pre-incubated with normal mouse serum (NMS) (Figure 9B). That the non-neutralizing mAh 4G2 escalated a sub-lethal DENV-2 S221 infection is consistent with its documented behavior (Watanabe S et al, Dengue virus infection with highly neutralizing levels of cross-reactive antibodies causes acute lethal small intestinal pathology without a high level of viremia in mice. J Virol. 2015; 89: 5847-5861). In contrast, the finding that anti-DENV-2 antiserum, despite fully neutralizing DENV-2 S221 (based on virus neutralization assay), caused rapid and complete mortality is surprising. This shows that cross-reactive enhancing Abs far outweigh the neutralizing Abs, in the anti-DENV-2 antiserum. However, replacing mAh 4G2/anti -DENV-2 antiserum with anti-rec ZIKV E MAP antiserum during pre-incubation, generated ICs (DENV-2 S221+anti-rec ZIKV MAP antiserum group) which were largely non-lethal to the mice (>80% survival), comparable to the survival of mice challenged with just the non-lethal dose of DENV-2 S221 (VC). Survival of mice in the‘DENV-2 S221+anti-rec ZIKV E MAP antiserum’ group was not significantly different (p=0.5282) from that of the VC group, but significantly higher (p=0.0095) than that of either the‘DENV-2 S221+mAb 4G2’ group or‘DENV-2 S221+anti-DENV-2 antiserum’ group. It has been reported that both live as well as inactivated ZIKVs induce murine Abs which manifest significant ADE of DENV-2 in the AG129 model (Watanabe S, et. al., Dengue and Zika virus serological cross-reactivity and their impact on pathogenesis in mice. J Infect Dis. 2019; 219: 223-233). In this context, the rec-ZIKV E MAPs, by virtue of their lack of DENV-2 enhancing potential, are unique in that they offer an inherent safety aspect.The finding that rec ZIKV E MAP -induced Abs do not escalate a sub-lethal DENV-2 infection is an unexpected and novel finding.

To find out whether there would be a subset of Abs in the polyclonal Ab repertoire induced by the rec ZIKV E MAPs that may potentially cause enhancement of ZIKV infection in vivo , C57BL/6 Stat2 ^/ mouse model was deployed (Figure 10A). This mouse was recently shown to be susceptible to ZIKV infection and manifest neurological symptoms with underlying virus spread to the central nervous system and gonads. Severe disease in this model, caused by ZIKVs of the African lineage, is associated weight loss, neurological symptoms including hunched back, fore limb and hind limb paralysis, withhigh mortality around 1 week post-challenge. On the other hand, ZIKVs of the Asian lineage cause delayed onset of morbidity and clinical signs and rarely cause death. These mice usually recover by ~2 weeks post-challenge (Tripathi et al, A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses. PLoSPathog. 2017; 13: el 006258). To test for possible ADE of ZIKV, groups («=4-5) of 5-6 week old C57BL/6 Stat2 ^/ mice were injected with NMS, anti-DENV-3 antiserum or anti-rec ZIKV E MAP antiserum (20 mΐ/mouse, i.p.) and challenged 2 hours later with ZIKV strain PRVABC59 (5xl0 ³ PFU/mouse, i.d.). These were monitored over 12 days post-ZIKV infection, for survival, clinical symptoms (fore- and hind-limb paralysis, hunched back etc) and weight loss(Figure 10, panels B-D). The data from this experiment showed that the ZIKV challenge in the presence of low levels of anti-DENV-3 antibodies resulted in increased mortality, reflecting enhancement of sub-lethal ZIKV infection to a lethal infection by anti-DENV-3 antibodies. Surprisingly, rec ZIKV E MAP-induced Abs did not affect survival of these ZIKV- challenged mice (test group) adversely in comparison to mice that were challenged with ZIKV, but had not received the anti-rec ZIKV E MAP antiserum (control group). Consistent with this, mice in both the control and test groups showed essentially similar profiles of clinical symptoms and weight loss from which they recovered by 2 weeks post-challenge. This leads to the conclusion that Abs elicited by rec ZIKV E MAPs may not be capable of enhancing ZIKV infection.

Flavivirus-nAbs are postulated to facilitate enhancement of the homologous flavivirus when they are at sub -neutralizing concentrations (Pierson TC et al, The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe. 2007; 1: 135-145). In the experiment in Figure 10, after passive transfer of 20 mΐ anti-rec ZIKV MAP antiserum (RNT ₅₀ ~200) into the mouse (blood volume -1.5 ml), the circulating ZIKV nAb titers would have undergone -75 -fold dilution (with RNT50 dropping to sub -neutralizing levels of -3). In contrast to that predicted by prior work (Pierson TC et al, The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe. 2007; 1: 135-145), this low level of ZIKV nAb titer did not promote enhancement of ZIKV infection upon challenge of the Stat2 ^/ mice. This leads to the important conclusion that ZIKV EDIII- directed antibodies elicited by rec ZIKV E MAPs do not enhance ZIKV infection at sub- neutralizing concentrations. This is a unique and novel outcome.