A single amino acid substitution in a recombinant G protein vaccine drastically curtails protective immunity against respiratory syncytial virus (RSV)

Recent studies have indicated a dominant T cell epitope located approximately between amino acids 184 and 203 on the respiratory syncytial virus (RSV) G protein. Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128-229) of the RSV G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids on vaccine efficacy. By changing two amino acids Arg 188 and Lys 192 to alanine, the ability of the Trx-G 128-229 fusion protein to protect mice against RSV challenge was virtually abolished. Mice immunized with the double mutant protein showed low levels of neutralizing antibodies and no pulmonary eosinophilic infiltrate, in contrast to that observed in mice immunized with wild type protein prior to RSV challenge. While less effective than the double mutant, mutation of either Arg 188 or Lys 192 to Ala drastically impaired the ability of immunized Trx-G 128-229 to induce neutralizing antibodies and to elicit pulmonary eosinophilia associated with RSV challenge. Despite low levels of virus-neutralizing antibodies, G protein-specific antibodies were detected by Western blotting in the sera from mice immunized with either of the single mutants (Arg 188 or Lys 192) but not the double mutant. Finally, immunization of mice with truncated forms of the Trx-G protein, showed partial protection against RSV challenge with Trx-G 128-188 but not with Trx-G 189-229. Taken together, the results indicate an important role for Arg 188 and Lys 192 in the induction of protective immunity and priming for eosinophilia against RSV. Furthermore, while the dominant protective linear epitope on the RSV G protein requires an intact sequence around Arg 188, there are additional, but less potent, protective epitopes upstream of Arg 188.     

Functional mapping of protective epitopes within the rotavirus VP6 protein in mice belonging to different haplotypes

We recently used "functional mapping" to locate protective epitopes in the carboxyl terminus (aa 197-397) of the VP6 protein (designated CD) of the EDIM strain of murine rotavirus [J. Virol. 74 (2000) 11574]. For this, H-2(d) BALB/c mice were given two intranasal (i.n.) immunizations (separated by 2 weeks) with VP6 or CD genetically-fused to maltose-binding protein, or with overlapping synthetic CD peptides, along with LT(R192G), a genetically-attenuated E. coli heat-labile toxin. The protective efficacies, i.e., percentage reductions in rotavirus shedding relative to control mice during 7 days following oral challenge with EDIM, were determined 4 weeks after the second immunization. Five of the 11 overlapping CD peptides stimulated significant protection (57-85%, P<0.05). Furthermore, chimeric VP6, the CD fragment, and a 14-amino-acid VP6 peptide within CD (RLSFQLMRPPNMTP), identified as a H-2(d)-restricted CD4 T cell epitope, were highly protective (93-98%, P<0.05). In this study, we continued to utilize functional mapping to show that the 14-mer peptide elicited significant protection (97.0%, P<0.05) in another H-2(d) mouse strain (DBA/2) but partial protection in H-2(b) 129 (39.2%) and C57Bl/6 (53.6%) as well as H-2(k) C3H (44.6%) mice. The first 13 amino acids of this 14-mer were necessary to induce maximal protection in H-2(d) mice. In addition, the H-2(b) 129 mice were immunized intranasally (i.n.) with 10 of the synthetic CD peptides and 5 were found to induce significant protection (90-97%, P<0.05). We also performed functional mapping to identify MHC class I epitopes in rotavirus proteins. A class I-binding epitope for H-2(b) C57Bl/6 mice had previously been mapped by ex vivo CTL assays within the VP6 protein and two additional class I epitopes were identified by computer-based prediction. When examined for their protective efficacies by functional mapping, two of the three were found to be partially but not significantly protective (44 and 46%, P>0.05). To better determine the usefulness of our in vivo methods to identify MHC class I-binding epitopes, four epitopes from the outer capsid VP7 rotavirus protein determined in ex vivo assays were evaluated for their protective efficacies and two were found to be partially protective. Together, these studies show that functional mapping is useful in locating epitopes that are relevant to the development of subunit rotavirus vaccines.     

Intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus

The oligonucleotides coding for three epitopes (HA91-108, NP55-69, and NP 147-158) of influenza virus, stimulating B-cells, T-helper cells and cytotoxic T lymphocytes (CTLs), respectively, were previously employed for expressing each epitope in flagella that induced specific humoral and cellular immune responses. We have constructed new plasmids expressing all three epitopes as a single recombinant product. Two versions have been prepared-a longer one (Fla-HNN) comprising hybrid flagella containing the epitopes, and a shorter version (HNN). Immunization of BALB/c mice with either constructs induced significant humoral immune response against influenza virus. The mice immunized with these peptides also induced higher T-helper activity, including Th1 type-cytokine (IL-2 and IFN-gamma) release. In addition, the mice immunized with HNN peptide demonstrated significant protection against sublethal viral challenge. Furthermore, this vaccine fully protected mice from lethal challenge and enhanced their recovery process. Our results indicate that a single construct expressing multiple epitopes, which stimulate different arms of the immune system, might be an appropriate candidate when the synthetic recombinant vaccine approach is considered.     

A hemagglutinin-derived peptide-vaccine ignored by virus-neutralizing passive antibodies, protects against murine measles encephalitis.

The neutralizing and protective monoclonal antibody BH47 defines the sequential epitope H236-255 of the measles virus hemagglutinin protein (MV-H). The objective of this study was to design peptides combining this B cell epitope (BCE) with different T cell epitopes (TCE) to obtain protective immunity. Most TTB peptides based on the 15mer BCE H236-250 induced MV-crossreactive antibodies, but only certain TCE induced virus neutralizing antibodies. The shortest BCE required for MV-reactivity and -neutralization was the 8mer H243-250 containing residue R243 implicated in CD46 down-regulation. Sera obtained after immunization with the TTB peptide containing the MV-derived TCE F421-435 protected mice against a lethal challenge with a neuro-adapted MV strain. Our results further demonstrate that this TTB peptide is fully immunogenic, even in the presence of protective levels of pre-existing MV-specific antibodies, suggesting that subunit vaccines based on such peptides could potentially be used to immunize infants in the presence of persisting maternal antibodies. It is therefore interesting that neutralizing antibodies were also obtained with a TTB peptide comprising a human promiscuous TCE (tt830). However, our results also emphasize the need to test sera induced with epitope-based vaccines against different virus strains, in particular if the epitope is not fully conserved.     

VennVax, a DNA-prime, peptide-boost multi-T-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone

The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming.     

Identification of protective and non-protective T cell epitopes in influenza

Understanding the immune response to different CD8 T cell epitopes is important for the development of vaccines designed to promote protective cellular immunity. Recently, we have shown that vaccination with the PA(224-233)/D(b) epitope of influenza virus was poorly protective in terms of viral clearance. To determine if other influenza virus epitopes behave in this manner, we analyzed the ability of three newly identified CD8 T cell epitopes and three previously defined epitopes to provide protection following vaccination and viral challenge. All six of the peptide-based vaccinations resulted in significantly increased numbers of epitope-specific CD8 T cells in the spleen. Interestingly, we found that vaccination with three peptides (HA(332-340), M1(128-135), or PA(224-233)) resulted in delayed viral clearance following infection. These findings indicate that some epitopes have a detrimental impact on viral clearance and have important implications for the development of vaccination strategies designed to provide protection against subsequent influenza virus challenge.     

Identification of B- and T-cell epitopes from glycoprotein B of herpes simplex virus 2 and evaluation of their immunogenicity and protection efficacy

Herpes simplex virus (HSV) infection is a major health concern worldwide. Evidence obtained from animals and humans indicates that B- and T-cell responses contribute to protective immunity against herpes virus infection. Glycoprotein B is a transmembrane envelope component of HSV-1 and HSV-2, which plays an important role in virion morphogenesis and penetration into host cells, and can induce neutralizing antibodies and protective T-cell response when it is used to immunize humans and animals. However, little is known about gB epitopes that are involved in B- and T-cell activities in vitro and in vivo. Thus, the HSV-2 gB sequence was screened using B- and T-cell epitope prediction systems, and the B-cell regions and the HLA-A*0201-restricted epitopes were identified. These B-cell epitopes elicited high IgG antibody titers in Balb/C mice, with a predominantly IgG1 subclass distribution, which indicated a Th2 bias. Specific IgGs induced by these two epitopes were evaluated as the neutralizing antibodies for virus neutralization. The predicted T-cell epitopes stabilized the HLA-A*0201 molecules on T(2) cells, and stimulate interferon-γ-secreting and cytotoxic CD8(+) T cells. Immunization with the predicted peptides reduced virus shedding and protected against lethal viral challenge in mice. The functional epitopes described herein, both B- and T-cell epitopes, are potentially implicated in vaccine development.     

Amino acid residues 196–225 of LcrV represent a plague protective epitope

LcrV, a protein that resides at the tip of the type III secretion needles of Yersinia pestis, is the single most important plague protective antigen. Earlier work reported monoclonal antibody MAb 7.3, which binds a conformational epitope of LcrV and protects experimental animals against lethal plague challenge. By screening monoclonal antibodies directed against LcrV for their ability to protect immunized mice against bubonic plague challenge, we examined here the possibility of additional protective epitopes. MAb BA5 protected animals against plague, neutralized the Y. pestis type III secretion pathway and promoted opsonophagocytic clearance of bacteria in blood. LcrV residues 196–225 were necessary and sufficient for MAb BA5 binding. Compared to full-length LcrV, a variant lacking its residues 196–225 retained the ability of eliciting plague protection. These results identify LcrV residues 196–225 as a linear epitope that is recognized by the murine immune system to confer plague protection.     

Candidate peptide vaccine induced protection against classical swine fever virus

Former investigations demonstrated that the envelope glycoprotein E2 could protect pigs from classical swine fever virus (CSFV). Based on these findings, we prepared synthetic peptide vaccine using E2 N-terminal antigenic units B/C and hoped to induce protective activity against lethal challenge of virulent CSFV strain Shimen. Five overlapped peptides sequence-covering amino acids 693-777 on E2 of Shimen were synthesized and then conjugated with bovine serum albumin (BSA), respectively. In the vaccination course, the candidate peptide vaccines in combination (multi-peptide vaccine (MPV)) were applied for immunization of pigs (n=10) and induced strong antibody response against CSFV. It is subsequently demonstrated that this peptide vaccine could provide immunized pigs complete protection against lethal CSFV challenge as C-strain does, while all non-immunized pigs in negative control group manifested obvious typical symptoms and died during the second and third weeks after viral challenge. In order to confirm the neutralizing activity of the polyclonal antibodies induced by MPV, neutralization assay were carried out on rabbits. The live C-strain alone could ordinarily induce typical fever on rabbits. The typical fever of rabbits induced by the live C-strain could be inhibited by pre-incubation with the anti-sera (dilution 1:4 and 1:16) induced by MPV, but not inhibited by pre-incubation with the same anti-sera from which the antibodies against five peptides were removed by peptide-specific affinity chromatography, which indicates that these peptide-specific antibodies in the anti-sera induced by MPV provided protective activity against CSFV. Our finding provides a new way to develop marker vaccine against CSFV.     

Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits

The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.     

Identification of conserved neutralizing linear epitopes within the VP1 protein of coxsackievirus A16

Coxsackievirus A16 (CA16) is a major causative agent of hand, foot, and mouth disease. Immunization with inactivated whole-virus or recombinant virus-like particles (VLP) of CA16 elicits neutralizing antibodies that protect mice against lethal challenge. However, the epitope/s responsible for this induction has not been determined. In this investigation, we identified six neutralizing linear epitopes of CA16. A panel of 95 synthetic peptides spanning the entire VP1 protein of CA16 were screened by ELISA for reactivity with neutralizing antisera against CA16 VLPs, which were generated in a previous study (Vaccine 30:6642–6648). Fifteen high-binding peptides were selected and further examined for their inhibitory effect on neutralization by anti-VLP sera. Among them, six peptides with no overlap significantly inhibited neutralization. Mice immunized with these six peptides generated peptide-specific serum antibodies. The anti-peptide antisera positively detected CA16 via immunofluorescent staining and Western blot assays. More importantly, they neutralized both homologous and heterologous CA16 strains, indicating that these six peptides represented neutralizing epitopes. Sequence alignment also showed that these epitopes are extremely conserved among CA16 strains of different genotypes. These findings have important implications for the development of peptide-based broadly protective CA16 vaccines.     

Candidate peptide-vaccines induced immunity against CSFV and identified sequential neutralizing determinants in antigenic domain A of glycoprotein E2

Antigenic domain A is a highly conserved unit on envelope protein E2 of classical swine fever virus (CSFV). It was found that mutant E2 containing only unit A, with the unit BC deleted, provided immunized pigs with complete protection against the lethal challenge. In this study, six overlapping peptides (A1-A6) covering this unit were synthesized and conjugated to bovine serum albumin (BSA). Two candidate multi-peptide-vaccines (MPVs) using aluminum adjuvant successfully induced potent immunity against CSFV in pigs. Although both candidate MPVs failed to provide complete protection, they showed better protective activity than that induced by C-strain. Subsequently, neutralizing epitopes in unit A were identified using a panel of peptide-vaccines (PVs). Six candidate peptide-vaccines (PV-An, n=1-6) were separately given to six groups of pigs. Among these candidates, PV-A2 and PV-A6 exhibited the most potent protective activity, while the other four showed weaker or almost no effects. Moreover, the polyclonal antibodies induced by PV-A2 and PV-A6 were capable of neutralizing C-strain virus at the dilution 1:16 in vitro. Thus, two principal sequential neutralizing determinants covered by peptide A2 (aa792-814) and A6 (aa844-865) were demonstrated to exist in the antigenic domain A, and can be recruited in developing new effective "marker vaccine" against CSFV.     

Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector

We expressed two immunogenic dominant epitopes of foot-and-mouth disease virus (FMDV) serotype O in tobacco plant using a vector based on a recombinant tobacco mosaic virus (TMV). The recombinant viruses TMVF11 and TMVF14 contained peptides of 11 and 14 amino acid residues, respectively, from FMDV VP 1 fused to the open reading frame of TMV coat protein (CP) gene between amino acid residues 154 and 155. TMVF11 and TMVF14 systemically infected tobacco plant and produced large quantities of stable progeny viral particles assembled with the modified CP subunits. Guinea pigs, mice and swine were used to test the protective effects of the recombinant viruses against FMDV infection. Most guinea pigs were protected against FMDV challenge after parenteral injection with TMVF11, TMVF14, or the mixture TMVF11/TMVF14, but not wtTMV. The TMVF11/TMVF14 mixture protected all animals when challenged with 150 guinea pig 50% infection dosage (GPID(50)) FMDV. Oral administration of the TMVF11/TMVF14 mixture (3mg total) protected 3/8 guinea pigs against the same FMDV challenge. Most of the suckling mice parenterally injected with antiserum from guinea pigs immunized with the TMVF11/TMVF14 mixture, but not with wtTMV, were also protected against FMDV challenge with 10 suckling mouse 50% lethal dosage (SMLD(50)), indicating that antibodies produced in guinea pigs immunized with the TMVF11/TMVF14 mixture specifically neutralized FMDV. Western blot analysis indicated that antiserum from those guinea pigs reacted with the FMDV VP1 protein. The protective effect of TMVF11 was also demonstrated in swine, where preliminary tests showed that nine pigs immunized with TMVF11 in three experiments were protected against FMDV challenge with 20 minimal infecting dose (MID).     

Addition of C3d-P28 adjuvant to a rabies DNA vaccine encoding the G5 linear epitope enhances the humoral immune response and confers protection

Rabies DNA vaccines based on full-length glycoprotein (G) induce virus neutralizing antibody (VNA) responses and protect against the virus challenge. Although conformational epitopes of G are the main target of VNAs, some studies have shown that a polypeptide linear epitope G5 is also able to induce VNAs. However, a G5 DNA vaccine has not been explored. While multiple doses of DNA vaccines are required in order to confer a protective immune response, this could be overcome by the inclusion of C3d-P28, a molecular adjuvant is know to improve the antibody response in several anti-viral vaccine models. To induce and enhance the immune response against rabies in mice, we evaluated two DNA vaccines based on the linear epitope G5 of Rabies Virus (RABV) glycoprotein (pVaxG5 vaccine) and another vaccine consisting of G5 fused to the molecular adjuvant C3d-P28 (pVaxF1 vaccine). VNA responses were measured in mice immunized with both vaccines. The VNA levels from the group immunized with pVaxG5 decreased gradually, while those from the group vaccinated with pVaxF1 remained high throughout the experimental study. After challenge with 22 LD50 of the Challenge Virus Strain (CVS), the survival rate of mice immunized with pVaxG5 and pVaxF1 was increased by 27% and 50% respectively, in comparison to the PBS group. Furthermore, the in vitro proliferation of anti-rabies specific spleen CD4+ and CD8+ T cells from mice immunized with pVaxF1 was observed. Collectively, these results suggest that the linear G5 epitope is a potential candidate vaccine. Furthermore, the addition of a C3d-P28 adjuvant contributed to enhanced protection, the sustained production of VNAs, and a specific T-cell proliferative response.     

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes.

Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible and that wild type influenza virus flanking amino acid sequences can influence the CTL response but are not essential for optimal CTL induction. We also examined the effect of different new amino acid sequences flanking the CTL epitopes. In one version, two CTL epitopes were linked together as 'string of beads'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu-induced CTL responses and tested for their protective effect against a lethal influenza A virus infection in mice and no effect was found. We conclude that a specific and highly directed CTL induction is possible by unlinked minigene DNA immunisation, but that CTL induction solely is not always sufficient to provide protection.     

Immunization with high epitope density of M2e derived from 2009 pandemic H1N1 elicits protective immunity in mice

As highly conserved amongst human influenza A strains, the extracellular domain of influenza M2 protein (M2e) is considered and proved as a promising candidate for a universal influenza vaccine. However, there are four amino acid variations in the M2e sequence of the 2009 pandemic H1N1 (referred to as "swine flu M2e, SFM2e") compared with the conventional M2e consensus sequence. Whether the sequence variation alters the immunogenicity and protection of SFM2e epitope remains unclear. In our present study, we synthesized SFM2e peptide and constructed a series of GST fusion proteins containing various copies of the SFM2e epitope and immunized mice to evaluate their immunogenicity and protective activity. We found that although the amino acid variations have weakened the immunogenicity of the SFM2e peptide, the SFM2e fusion proteins with high epitope densities induced intense and diverse antibody response as well as T cell response. Moreover, mice immunized with high epitope density of SFM2e were nearly fully protected against a lethal challenge by the mouse-adapted influenza virus A/Porto Rico/8/34. Our study could provide new available data to improve the epitope vaccine strategy against influenza pandemic.     

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.     

Protection against hydatid disease induced with the EG95 vaccine is associated with conformational epitopes

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.     

Immunization with a consensus epitope from human papillomavirus L2 induces antibodies that are broadly neutralizing

Vaccines targeting conserved epitopes in the HPV minor capsid protein, L2, can elicit antibodies that can protect against a broad spectrum of HPV types that are associated with cervical cancer and other HPV malignancies. Thus, L2 vaccines have been explored as alternatives to the current HPV vaccines, which are largely type-specific. In this study we assessed the immunogenicity of peptides spanning the N-terminal domain of L2 linked to the surface of a highly immunogenic bacteriophage virus-like particle (VLP) platform. Although all of the HPV16 L2 peptide-displaying VLPs elicited high-titer anti-peptide antibody responses, only a subset of the immunogens elicited antibody responses that were strongly protective from HPV16 pseudovirus (PsV) infection in a mouse genital challenge model. One of these peptides, mapping to HPV16 L2 amino acids 65–85, strongly neutralized HPV16 PsV but showed little ability to cross-neutralize other high-risk HPV types. In an attempt to broaden the protection generated through vaccination with this peptide, we immunized mice with VLPs displaying a peptide that represented a consensus sequence from high-risk and other HPV types. Vaccinated mice produced antibodies with broad, high-titer neutralizing activity against all of the HPV types that we tested. Therefore, immunization with virus-like particles displaying a consensus HPV sequence is an effective method to broaden neutralizing antibody responses against a type-specific epitope.     

Development of a novel ZIKV vaccine comprised of immunodominant CD4+ and CD8+ T cell epitopes identified through comprehensive epitope mapping in Zika virus infected mice

Zika virus (ZIKV) caused over two million human infections in more than 80 countries around 2015–2016. Current vaccines under development are mostly focused on inducing antibodies that despite capable of inhibiting the virus, may have the potential to trigger antibody dependent enhancement (ADE). T cell vaccines that do not induce antibodies targeting viral surface will unlikely cause ADE, but be capable of potentiating the effectiveness of an antibody-inducing vaccine. To develop such a protective T cell vaccine, we first examined the repertoire of antigen-specific T cells in immunocompetent mice that have been transiently infected by ZIKV. Through epitope mapping using 427 overlapping peptides spanning the entire length of ZIKV polyprotein, we discovered 27 immunodominant epitopes scattered throughout the virus on C, E, NS1-NS5 proteins. Among them, 8 were confirmed as CD4+ T cell epitopes, and 16 as CD8+ T cell epitopes, while 3 for both T cell subsets. From these 27 newly identified epitopes, the top 10 epitopes were selected to formulate three T cell vaccines comprised of either CD4+ T cell epitopes, or CD8+ T cell epitopes, or a mixture of both. Immunization with these T cell epitopes induced T cell-mediated cytotoxicity and cytokine production, and conferred varying degrees of protection against ZIKV challenge. Moreover, these new T cell vaccines also improved the protective efficacy of a neutralizing antibody-inducing recombinant E80 protein vaccine. Together, our results provided additional evidence in support of the protective role of ZIKV-specific CD4+ and CD8+ T cells, and laid foundation for future development of T cell vaccines for ZIKV.