Sublingual Immunization of Trivalent Human Papillomavirus DNA Vaccine in Baculovirus Nanovector for Protection against Vaginal Challenge

Here, we report the immunogenicity of a sublingually delivered, trivalent human papillomavirus (HPV) DNA vaccine encapsidated in a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus nanovector. The HERV envelope-coated, nonreplicable, baculovirus-based DNA vaccine, encoding HPV16L1, -18L1 and -58L1 (AcHERV-triHPV), was constructed and sublingually administered to mice without adjuvant. Following sublingual (SL) administration, AcHERV-triHPV was absorbed and distributed throughout the body. At 15 minutes and 1 day post-dose, the distribution of AcHERV-triHPV to the lung was higher than that to other tissues. At 30 days post-dose, the levels of AcHERV-triHPV had diminished throughout the body. Six weeks after the first of three doses, 1×10⁸ copies of SL AcHERV-triHPV induced HPV type-specific serum IgG and neutralizing antibodies to a degree comparable to that of IM immunization with 1×10⁹ copies. AcHERV-triHPV induced HPV type-specific vaginal IgA titers in a dose-dependent manner. SL immunization with 1×10¹⁰ copies of AcHERV-triHPV induced Th1 and Th2 cellular responses comparable to IM immunization with 1×10⁹ copies. Molecular imaging revealed that SL AcHERV-triHPV in mice provided complete protection against vaginal challenge with HPV16, HPV18, and HPV58 pseudoviruses. These results support the potential of SL immunization using multivalent DNA vaccine in baculovirus nanovector for induction of mucosal, systemic, and cellular immune responses.     

Multivalent Human Papillomavirus L1 DNA Vaccination Utilizing Electroporation

Objectives

Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV) L1 and L2 DNA vaccination.

Methods

Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11−88 of 8 different HPV types (11−88×8) or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.

Results

Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2) vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type.

Discussion

Multivalent vaccination via in vivo electroporation requires spatial separation of individual type L1 DNA vaccines.     

Relationship between Humoral Immune Responses against HPV16, HPV18, HPV31 and HPV45 in 12-15 Year Old Girls Receiving Cervarix? or Gardasil? Vaccine

Background

Human papillomavirus (HPV) vaccines confer protection against the oncogenic genotypes HPV16 and HPV18 through the generation of type-specific neutralizing antibodies raised against virus-like particles (VLP) representing these genotypes. The vaccines also confer a degree of cross-protection against HPV31 and HPV45, which are genetically-related to the vaccine types HPV16 and HPV18, respectively, although the mechanism is less certain. There are a number of humoral immune measures that have been examined in relation to the HPV vaccines, including VLP binding, pseudovirus neutralization and the enumeration of memory B cells. While the specificity of responses generated against the vaccine genotypes are fairly well studied, the relationship between these measures in relation to non-vaccine genotypes is less certain.

Methods

We carried out a comparative study of these immune measures against vaccine and non-vaccine genotypes using samples collected from 12–15 year old girls following immunization with three doses of either Cervarix^® or Gardasil^® HPV vaccine.

Results

The relationship between neutralizing and binding antibody titers and HPV-specific memory B cell levels for the vaccine genotypes, HPV16 and HPV18, were very good. The proportion of responders approached 100% for both vaccines while the magnitude of these responses induced by Cervarix^® were generally higher than those following Gardasil^® immunization. A similar pattern was found for the non-vaccine genotype HPV31, albeit at a lower magnitude compared to its genetically-related vaccine genotype, HPV16. However, both the enumeration of memory B cells and VLP binding responses against HPV45 were poorly related to its neutralizing antibody responses. Purified IgG derived from memory B cells demonstrated specificities similar to those found in the serum, including the capacity to neutralize HPV pseudoviruses.

Conclusions

These data suggest that pseudovirus neutralization should be used as the preferred humoral immune measure for studying HPV vaccine responses, particularly for non-vaccine genotypes.     

A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2

Background

Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin.

Methodology/Principal Findings

L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV.

Conclusion/Significance

VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.     

Enhancement of vaccine potency by fusing modified LTK63 into human papillomavirus type 16 chimeric virus-like particles

To enhance the immunogenicity of human papillomavirus 16 (HPV 16) virus-like particles (VLPs), the modified adjuvant, mLTK63, was fused to the C-terminus of HPV 16 L2 protein. Coexpression of HPV 16 L1 and L2-mLTK63 proteins in insect cells led to the efficient assembly of HPV 16 L1/L2-mLTK63 chimeric VLPs (cVLPs), which combined the antigen and adjuvant as a unit. Compared with HPV 16 L1/L2 VLPs, the HPV 16 L1/L2-mLTK63 cVLPs had similar structural biology characteristics and binding activities with the cell surface receptors and HPV 16-specific neutralizing monoclonal antibodies. Intramuscular immunization of BALB/c mice with the HPV 16 L1/L2-mLTK63 cVLPs could induce higher titers of HPV 16-specific long-lasting neutralizing serum antibodies and stronger splenocyte proliferation, Th1- and Th2-type cytokines and CD4(+) Th responses than HPV 16 L1/L2 VLPs. The results suggested that it is possible to enhance the immunogenicity of HPV VLP vaccines via a strategy of fusing effective adjuvant protein into cVLPs.     

A Chimeric 18L1-45RG1 Virus-Like Particle Vaccine Cross-Protects against Oncogenic Alpha-7 Human Papillomavirus Types

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV.     

A protective and broadly cross-neutralizing epitope of human papillomavirus L2

We generated a monoclonal antibody, RG-1, that binds to highly conserved L2 residues 17 to 36 and neutralizes human papillomavirus 16 (HPV16) and HPV18. Passive immunotherapy with RG-1 was protective in mice. Antiserum to the HPV16 L2 peptide comprising residues 17 to 36 (peptide 17-36) neutralized pseudoviruses HPV5, HPV6, HPV16, HPV 18, HPV31, HPV 45, HPV 52, HPV 58, bovine papillomavirus 1, and HPV11 native virions. Depletion of HPV16 L2 peptide 17-36-reactive antibodies from cross-neutralizing rabbit and human L2-specific sera abolished cross-neutralization and drastically reduced neutralization of the cognate type. This cross-neutralization of diverse HPVs associated with cervical cancer, genital warts, and epidermodysplasia verruciformis suggests the possibility of a broadly protective, peptide-based vaccine.     

Human papillomavirus type 16 L1 capsomeres induce L1-specific cytotoxic T lymphocytes and tumor regression in C57BL/6 mice

We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis ((51)Cr release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D(b)-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and (51)Cr release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1 Delta N10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 mice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose-dependent manner as measured by ELISPOT and (51)Cr release assay. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins.     

Identification of Broad-Genotype HPV L2 Neutralization Site for Pan-HPV Vaccine Development by a Cross-Neutralizing Antibody

Human Papillomavirus (HPV), a non-enveloped, double-stranded DNA virus, is responsible for 5% of human cancers. The HPV capsid consists of major and minor structural proteins, L1 and L2. L1 proteins form an icosahedral shell with building blocks of the pentameric capsomere, and one L2 molecule extends outward from the central hole of the capsid. Thus, L2 is concealed within L1 and only becomes exposed when the capsid interacts with host cells. The low antigenic variation of L2 means that this protein could offer a target for the development of a pan-HPV vaccine. Toward this goal, here we describe an anti-L2 monoclonal antibody, 14H6, which broadly neutralizes at least 11 types of HPV, covering types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58 and 59, in pseudovirion—based cell neutralization assay. The mAb 14H6 recognizes a minimal linear epitope located on amino acids 21 to 30 of the L2 protein. Alanine scanning mutagenesis and sequence alignment identified several conserved residues (Cys22, Lys23, Thr27, Cys28 and Pro29) that are involved in the 14H6 binding with L2. The epitope was grafted to several scaffolding proteins, including HPV16 L1 virus-like particles, HBV 149 core antigen and CRM197. The resultant chimeric constructs were expressed in Escherichia coli and purified with high efficiency. Immunization with these pan-HPV vaccine candidates elicited high titers of the L2-specific antibody in mice and conferred robust (3-log) titers of cross-genotype neutralization, including against HPV11, 16, 18, 45, 52, 58 and 59. These findings will help in the development of an L2-based, pan-HPV vaccine.     

Intranasal vaccination with recombinant adeno-associated virus type 5 against human papillomavirus type 16 L1

Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy in many preclinical studies, as well as in clinical trials. However, only a few approaches have used recombinant AAV (rAAV) to deliver vaccine antigens. We generated an rAAV encoding the major capsid protein L1 (L1h) from the human papillomavirus type 16 (HPV16), aiming to develop a prophylactic vaccine against HPV16 infections, which are the major cause of cervical cancer in women worldwide. A single dose of rAAV5 L1h administered intranasally was sufficient to induce high titers of L1-specific serum antibodies, as well as mucosal antibodies in vaginal washes. Seroconversion was maintained for at least 1 year. In addition, a cellular immune response was still detectable 60 weeks after immunization. Furthermore, lyophilized rAAV5 L1h successfully evoked a systemic and mucosal immune response in mice. These data clearly show the efficacy of a single-dose intranasal immunization against HPV16 based on the recombinant rAAV5L1h vector without the need of an adjuvant.     

Protection of rabbits against challenge with rabbit papillomaviruses by immunization with the N terminus of human papillomavirus type 16 minor capsid antigen L2

Current L1 virus-like particle (VLP) vaccines provide type-restricted protection against a small subset of the human papillomavirus (HPV) genotypes associated with cervical cancer, necessitating continued cytologic screening of vaccinees. Cervical cancer is most problematic in countries that lack the resources for screening or highly multivalent HPV VLP vaccines, suggesting the need for a low-cost, broadly protective vaccinogen. Here, N-terminal L2 polypeptides comprising residues 1 to 88 or 11 to 200 derived from HPV16, bovine papillomavirus type 1 (BPV1), or cottontail rabbit papillomavirus (CRPV) were produced in bacteria. Rabbits were immunized with these N-terminal L2 polypeptides and concurrently challenged with CRPV and rabbit oral papillomavirus (ROPV). Vaccination with either N-terminal L2 polypeptides of CRPV effectively protected rabbits from CRPV challenge but not from papillomas induced by cutaneous challenge with CRPV genomic DNA. Furthermore, papillomas induced by CRPV genomic DNA deficient for L2 expression grew at the same rate as those induced by wild-type CRPV genomic DNA, further suggesting that the L2 polypeptide vaccines lack therapeutic activity. Neutralizing serum antibody titers of >15 correlated with protection (P < 0.001), a finding consistent with neutralizing antibody-mediated protection. Surprisingly, a remarkable degree of protection against heterologous papillomavirus types was observed after vaccination with N-terminal L2 polypeptides. Notably, vaccination with HPV16 L2 11-200 protected against cutaneous and mucosal challenge with CRPV and ROPV, respectively, papillomaviruses that are evolutionarily divergent from HPV16. Further, vaccination with HPV16 L2 11-200 generates broadly cross-neutralizing serum antibody, suggesting the potential of L2 as a second-generation preventive HPV vaccine antigen.     

Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

Background

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/Principal Findings

In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4⁺ and CD8⁺ T cells, with the CD8⁺ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4⁺ and CD8⁺ T-cell immune responses. HIV-1-specific CD4⁺ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8⁺ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8⁺ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/Significance

These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.     

Development of AAVLP(HPV16/31L2) particles as broadly protective HPV vaccine candidate

The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17-36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2--simulating the high prevalence of AAV2 antibodies in the population--even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected na?ve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.     

人乳头瘤病毒16型-L1真核表达质粒的构建和表达以及实验动物对裸DNA的免疫反应

Recombinant plasmid pcDNA-L1 was constructed by inserting HPV16-L1 gene fragment into eukaryotic expression plasmid pcDNA3.1. pcDNA-L1 was transfected into mammalian cells Cos-7 and the expression of HPV16-L1 protein was testified by immunohistochemistry(IHC). Then the recombinant plasmid was directly injected into the quadriceps muscle of BALB/c mice. Antibodies against HPV16-L1 in the immunized mice were positively detected by immunodot and IHC at 28 and 41 days after the last immunization.     

Study of cell-mediated response in mice by HPV16 L1 virus-like particles expressed in Saccharomyces cerevisiae

The first vaccine against human papillomaviruses (HPV) formulated with HPV16 L1 virus-like particles (VLPs) produced in yeast was approved by the FDA in June 2006. Nevertheless, there have been few studies of the immunogenicity in mice of VLPs. In this study, we evaluated the cell-mediated immune response to VLPs produced in Saccharomyces cerevisiae. After immunization of mice with HPV16 L1 VLPs, we measured splenocytes proliferation and the levels of IFNgamma, IL2, IL4, and IL5. Splenocytes proliferation was significantly increased and a mixed Th1/Th2 response was indicated. IgG subtype immunoresponses were strongly induced and IgG1 titers were higher than those of IgG2a.     

A Vaccine of L2 Epitope Repeats Fused with a Modified IgG1 Fc Induced Cross-Neutralizing Antibodies and Protective Immunity against Divergent Human Papillomavirus Types

Current human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs)-based vaccines in clinic induce strong HPV type-specific neutralizing antibody responses. To develop pan-HPV vaccines, here, we show that the fusion protein E3R4 consisting of three repeats of HPV16 L2 aa 17–36 epitope (E3) and a modified human IgG1 Fc scaffold (R4) induces cross-neutralizing antibodies and protective immunity against divergent HPV types. E3R4 was expressed as a secreted protein in baculovirus expression system and could be simply purified by one step Protein A affinity chromatography with the purity above 90%. Vaccination of E3R4 formulated with Freunds adjuvant not only induced cross-neutralizing antibodies against HPV pseudovirus types 16, 18, 45, 52, 58, 6, 11 and 5 in mice, but also protected mice against vaginal challenges with HPV pseudovirus types 16, 45, 52, 58, 11 and 5 for at least eleven months after the first immunization. Moreover, vaccination of E3R4 formulated with FDA approved adjuvant alum plus monophosphoryl lipid A also induced cross-neutralizing antibodies against HPV types 16, 18 and 6 in rabbits. Thus, our results demonstrate that delivery of L2 antigen as a modified Fc-fusion protein may facilitate pan-HPV vaccine development.     

Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund''s adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund''s adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.The more than 100 types of human papillomaviruses (HPV) identified to date (14) are the etiological agents of skin and mucosal papillomas or warts. Persistent infection with high-risk mucosal types, most often HPV type 16 (HPV16) and HPV18, causes cervical cancer, which constitutes the second leading fatal cancer in women worldwide, causing 274,000 deaths per year. Substantial morbidity results from other noncervical HPV-related conditions, such as anogenital warts or anal cancer (23).The development of current prophylactic papillomavirus vaccines was launched by observations that recombinantly expressed major capsid protein L1 self-assembles into virus-like particles (VLP). These empty viral capsids are composed of 360 L1 molecules and resemble native virions in both structure and immunogenicity, yet are nononcogenic and noninfectious. Moreover, VLP cannot replicate because the cells in which VLP are made contain only L1 and no other papillomavirus genes. Subunit VLP vaccines induce high-titer and type-restricted antibody responses to conformational L1 epitopes (12, 26, 39, 44). When applied to women prior to infection, available vaccines targeting the most prevalent high-risk types, HPV16 and HPV18, have demonstrated up to 100% efficacy against persistent infection and associated disease caused by the included types and thus are potentially able to prevent ∼70% of cervical high-grade dysplasias and probably cancers (22, 46). Therefore, use of currently licensed L1 vaccines necessitates continuation of cytological cervical screening of women. The prevention of 96% of cervical cancer would require immunity to seven high-risk HPV types (HPV16, -18, -31, -33, -45, -52, and -58) (32) and the development of more highly multivalent (and presumably costly) L1 VLP vaccines.The search for alternative broader-spectrum immunogens drew attention to the minor capsid protein L2, which is immunogenically subdominant in the context of coexpressed L1-L2 capsids (38). Immunization of animals with the amino (N)-terminal peptide of L2 demonstrated its ability to elicit low-titer neutralizing antibodies that protect against challenge with cognate papillomavirus types in vivo (16, 19), cross-neutralize heterologous types in vitro (25, 33, 38), and confer cross-protection in vivo (17).This study addresses two major issues that may further the development of L2-based broader-spectrum vaccines. First, the N terminus of L2 is more closely examined for potential neutralization epitopes, by incorporating peptides into papillomavirus VLP as peptide-presenting platforms (7, 21, 42). Moreover, we take advantage of the immunogenic characteristics of virion surfaces, such as the dense repetitive surface array of VLP, to induce strong and enduring immune responses to displayed L2 epitopes.     

Skin vaccination against cervical cancer associated human papillomavirus with a novel micro-projection array in a mouse model

Background

Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer.

Methodology/Principal Findings

Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point.

Conclusions/Significance

Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.     

A live-attenuated HSV-2 ICP0 virus elicits 10 to 100 times greater protection against genital herpes than a glycoprotein D subunit vaccine

Glycoprotein D (gD-2) is the entry receptor of herpes simplex virus 2 (HSV-2), and is the immunogen in the pharmaceutical industry''s lead HSV-2 vaccine candidate. Efforts to prevent genital herpes using gD-2 subunit vaccines have been ongoing for 20 years at a cost in excess of $100 million. To date, gD-2 vaccines have yielded equivocal protection in clinical trials. Therefore, using a small animal model, we sought to determine if a live-attenuated HSV-2 ICP0 ⁻ virus would elicit better protection against genital herpes than a gD-2 subunit vaccine. Mice immunized with gD-2 and a potent adjuvant (alum+monophosphoryl lipid A) produced high titers of gD-2 antibody. While gD-2-immunized mice possessed significant resistance to HSV-2, only 3 of 45 gD-2-immunized mice survived an overwhelming challenge of the vagina or eyes with wild-type HSV-2 (MS strain). In contrast, 114 of 115 mice immunized with a live HSV-2 ICP0 ⁻ virus, 0ΔNLS, survived the same HSV-2 MS challenges. Likewise, 0ΔNLS-immunized mice shed an average 125-fold less HSV-2 MS challenge virus per vagina relative to gD-2-immunized mice. In vivo imaging demonstrated that a luciferase-expressing HSV-2 challenge virus failed to establish a detectable infection in 0ΔNLS-immunized mice, whereas the same virus readily infected naïve and gD-2-immunized mice. Collectively, these results suggest that a HSV-2 vaccine might be more likely to prevent genital herpes if it contained a live-attenuated HSV-2 virus rather than a single HSV-2 protein.     

Immunogenicity of a Trivalent Human Papillomavirus L1 DNA-Encapsidated,Non-Replicable Baculovirus Nanovaccine

Previously, we developed a non-replicating recombinant baculovirus coated with human endogenous retrovirus envelope protein (AcHERV) for enhanced cellular delivery of human papillomavirus (HPV) 16L1 DNA. Here, we report the immunogenicity of an AcHERV-based multivalent HPV nanovaccine in which the L1 segments of HPV 16, 18, and 58 genes were inserted into a single baculovirus genome of AcHERV. To test whether gene expression levels were affected by the order of HPV L1 gene insertion, we compared the efficacy of bivalent AcHERV vaccines with the HPV 16L1 gene inserted ahead of the 18L1 gene (AcHERV-HP16/18L1) with that of AcHERV with the HPV 18L1 gene inserted ahead of the 16L1 gene (AcHERV-HP18/16L1). Regardless of the order, the bivalent AcHERV DNA vaccines retained the immunogenicity of monovalent AcHERV-HP16L1 and AcHERV-HP18L1 DNA vaccines. Moreover, the immunogenicity of bivalent AcHERV-HP16/18L1 was not significantly different from that of AcHERV-HP18/16L1. In challenge tests, both bivalent vaccines provided complete protection against HPV 16 and 18 pseudotype viruses. Extending these results, we found that a trivalent AcHERV nanovaccine encoding HPV 16L1, 18L1, and 58L1 genes (AcHERV-HP16/18/58L1) provided high levels of humoral and cellular immunogenicity against all three subtypes. Moreover, mice immunized with the trivalent AcHERV-based nanovaccine were protected from challenge with HPV 16, 18, and 58 pseudotype viruses. These results suggest that trivalent AcHERV-HPV16/18/58L1 could serve as a potential prophylactic baculoviral nanovaccine against concurrent infection with HPV 16, 18, and 58.