Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine.     

Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies

Two conserved epitopes, located in the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41, are recognized by two HIV-1 broadly neutralizing antibodies 2F5 and 4E10, and are promising targets for vaccine design in efforts to elicit anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 or 4E10 epitope or the MPER domain into D-E loop of BPV L1 respectively. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting single epitopes. Oral immunization of mice with the chimeric VLPs displaying the 2F5 epitope or MPER domain elicited epitope-specific serum IgGs and mucosal secretory IgAs. The induced antibodies specifically recognized the native conformation of MPER in the context of HIV-1 envelope protein. The antibodies induced by chimeric VLPs presenting MPER domain are able to partially neutralize HIV-1 viruses from clade B and clade C.     

Hepatitis B virus-like particles expressing Plasmodium falciparum epitopes induce complement-fixing antibodies against the circumsporozoite protein

The repetitive structure of compact virus-like particles (VLPs) provides high density displays of antigenic sequences, which trigger key parts of the immune system. The hepatitis B virus (HBV) and human papilloma virus (HPV) vaccines exploit the assembly competence of structural proteins, which are the effective immunogenic components of the prophylactic HBV and HPV vaccines, respectively. To optimize vaccine designs and to promote immune responses against protective epitopes, the “Asp-Ala-Asp-Pro” (NANP)-repeat from the Plasmodium falciparum circumsporozoite protein (CSP) was expressed within the exposed, main antigenic site of the small HBV envelope protein (HBsAgS); this differs from the RTS,S vaccine, in which CSP epitopes are fused to the N-terminus of HBsAgS. The chimeric HBsAgS proteins are assembly competent, produce VLPs, and provide a high antigenic density of the NANP repeat sequence. Chimeric VLPs with four or nine NANP-repeats (NANP4 and NANP9, respectively) were expressed in mammalian cells, the HBsAgS- and CSP-specific antigenicity of the VLPs was determined, and the immunogenicity of the VLPs assessed in relation to the induction of anti-HBsAgS and anti-CSP antibody responses. The chimeric VLPs induced high anti-CSP titres in BALB/c mice independent of the number of the NANP repeats. However, the number of NANP repeats influenced the activity of vaccine-induced antibodies measured by complement fixation to CSP, one of the proposed effector mechanisms for Plasmodium neutralization in vivo. Sera from mice immunized with VLPs containing nine NANP repeats performed better in the complement fixation assay than the group with four NANP repeats. The effect of the epitope-specific density on the antibody quality may instruct VLP platform designs to optimize immunological outcomes and vaccine efficacy.     

Generation and immunogenicity of porcine circovirus type 2 chimeric virus-like particles displaying porcine reproductive and respiratory syndrome virus GP5 epitope B

Virus-like particles (VLPs) can be used as transfer vehicles carrying foreign proteins or antigen epitopes to produce chimeric VLPs for bivalent or multivalent vaccines. Based on the crystal structure of porcine circovirus type 2 (PCV2) capsid protein (Cap), in addition to alignment of the Cap sequences collected from various isolates of PCV2 and PCV1, we predicted that Loop CD of the PCV2 Cap should tolerate insertion of foreign epitopes, and furthermore that such an insertion could be presented on the surface of PCV2 VLPs. To validate this, the GP5 epitope B of porcine reproductive and respiratory syndrome virus (PRRSV) was inserted into Loop CD of the PCV2 Cap. The 3D structure of the recombinant PCV2 Cap (rCap) was simulated by homology modeling; it appeared that the GP5 epitope B was folded as a relatively independent unit, separated from the PCV2 Cap backbone. Furthermore, based on transmission electron microscopy, the purified PCV2 rCap self-assembled into chimeric VLPs which entered PK-15 cells. In addition, PCV2 chimeric VLPs induced strong humoral (neutralizing antibodies against PCV2 and PRRSV) and cellular immune responses in mice. We concluded that the identified insertion site in the PCV2 Cap had great potential to develop PCV2 VLPs-based bivalent or multivalent vaccines; furthermore, it would also facilitate development of a nano-device to present a functional peptide on the surface of the VLPs that could be used for therapeutic purposes.     

Hepatitis B virus core particles containing multiple epitopes confer protection against enterovirus 71 and coxsackievirus A16 infection in mice

Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two major causative agents of hand-foot-and-mouth disease (HFMD). To investigate novel combined vaccines to prevent EV71 and CA16 infection, we constructed chimeric virus-like particles (tHBc/SPA or tHBc/SP VLPs) displaying conserved epitopes of EV71 (aa 208–222 of VP1 and aa 248–263 of VP2) and CA16 (aa271-285 of VP1) using a truncated hepatitis B virus core carrier (tHBc). Immunization with the chimeric VLPs induced epitope- or virus-specific IgG and neutralization antibodies against EV71 and CA16 in the mice. Compared with inactivated EV71, the chimeric VLPs induced significantly increased Th1 cytokine (IFN-γ, IL-2) production and decreased Th2 cytokine (IL-4, IL-10) responses. Neonatal mice born to dams immunized with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection. Co-expression of the conserved human MHC class I CD4+ T cell epitope (aa248-263 of VP2) did not improve the antiviral immunity of the chimeric VLP vaccine in mice. Our results demonstrate that experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.     

Recombinant virus-like particles as a carrier of B- and T-cell epitopes of hepatitis C virus (HCV)

The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3' terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete "mature" VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants of hybrid particles induced high humoral and cellular responses to HBcAg. Immunization with the HBc/HCV core particles led to relatively low antibody and T-cell proliferative responses to HCV core epitopes. The HBc/HCV NS3 particles were able to induce high levels of anti-NS3 antibodies in the absence of proliferative responses to HCV epitopes. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCV-specific immunogen.     

Chimeric coronavirus-like particles carrying severe acute respiratory syndrome coronavirus (SCoV) S protein protect mice against challenge with SCoV

We tested the efficacy of coronavirus-like particles (VLPs) for protecting mice against severe acute respiratory syndrome coronavirus (SCoV) infection. Coexpression of SCoV S protein and E, M and N proteins of mouse hepatitis virus in 293T or CHO cells resulted in the efficient production of chimeric VLPs carrying SCoV S protein. Balb/c mice inoculated with a mixture of chimeric VLPs and alum twice at an interval of four weeks were protected from SCoV challenge, as indicated by the absence of infectious virus in the lungs. The same groups of mice had high levels of SCoV-specific neutralizing antibodies, while mice in the negative control groups, which were not immunized with chimeric VLPs, failed to manifest neutralizing antibodies, suggesting that SCoV-specific neutralizing antibodies are important for the suppression of viral replication within the lungs. Despite some differences in the cellular composition of inflammatory infiltrates, we did not observe any overt lung pathology in the chimeric-VLP-treated mice, when compared to the negative control mice. Our results show that chimeric VLP can be an effective vaccine strategy against SCoV infection.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

Recombinant retrovirus-derived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice

While the immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood, it is now admitted that an effective vaccine against HCV will need to induce both cellular and humoral immune responses and address viral heterogeneity to prevent immune escape. We developed a vaccine platform specifically aimed at inducing such responses against HCV antigens displayed by recombinant retrovirus-based virus-like particles (VLPs) made of Gag of murine leukemia virus. Both ex vivo produced VLPs and plasmid DNA encoding VLPs can be used as vaccines. Here, we report that immunizations with plasmid DNA forming VLPs pseudotyped with HCV E1 and E2 envelope glycoproteins (HCV-specific plasmo-retroVLPs) induce strong T-cell-mediated immune responses that can be optimized by using proper DNA delivery methods and/or genetic adjuvants. Additionally, multigenotype or multi-specific T-cell responses were observed after immunization with plasmids that encode VLPs pseudotyped with E1E2 derived from numerous viral genotypes and/or displaying NS3 antigen in capsid proteins. While homologous prime-boost immunizations with HCV-specific plasmo-retroVLPs or ex vivo produced VLPs induce a low level of specific antibody responses, optimal combination of plasmo-retroVLPs and VLPs was identified for inducing HCV-specific T-cell and B-cell responses as well as neutralizing antibodies. Altogether, these results have important meanings for the development of anti-HCV preventive vaccines and exemplify the flexibility and potential of our retrovirus-based platform in inducing broad cellular and humoral immune responses.     

Combination of three virus-derived nanoparticles as a vaccine against enteric pathogens; enterovirus,norovirus and rotavirus

Enteric viruses cause diverse infections with substantial morbidity and mortality in children, rotavirus (RV) and norovirus (NoV) being the leading agents of severe pediatric gastroenteritis. Coxsackie B viruses (CVB) are common enteroviruses (EV), associated with increased incidence of severe neonatal CVB disease with potentially fatal consequences. To prevent majority of childhood gastroenteritis, we have developed a non-live NoV–RV combination vaccine consisting of NoV virus-like particles (VLPs) and RV oligomeric rVP6 protein that induced protective immune responses to NoV and RV in mice. Moreover, rVP6 acted as an adjuvant for NoV VLPs. Here, we investigated a possibility to include a third enteric virus-derived antigen in the candidate NoV–RV vaccine, by adding recombinant nanoparticles derived from EV CVB1. To examine immunogenicity of EV-NoV-RV vaccine, BALB/c mice were immunized intramuscularly twice with 10 µg CVB1 VLPs, GII.4 VLPs and rVP6 nanotubes, either separately or combined. To evaluate the adjuvant effect of rVP6 on EV responses, mice received 0.3 µg CVB1 VLPs with or without 10 µg rVP6. Comparable serum IgG antibodies were detected whether the antigens were administered separately or in combination. Each formulation generated IgG1 and IgG2a antibodies, indicating a mixed Th2/Th1-type response. CVB1 VLPs skewed the isotype distribution slightly towards IgG1 subtype, while EV-NoV-RV combination vaccine induced unbiased Th1/Th2 responses to CVB1. Each antigen also induced T cell mediated immunity measured by IFN-γ secretion to specific stimulants ex vivo. Antisera raised by single antigens and combined formulation also exhibited strong neutralizing ability against CVB1 and NoV GII.4. Further, rVP6 showed an adjuvant effect on CVB1 responses, sparing the VLP dose and homogenizing the responses. Finally, the results support inclusion of additional antigens in the candidate NoV-RV combination vaccine to combat severe childhood infections and confirm adjuvant effect of rVP6 nanostructures.     

Virus-like particle vaccine induces cross-protection against human metapneumovirus infections in mice

Human metapneumovirus (HMPV) is a paramyxovirus that causes acute respiratory-tract infections in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We engineered HMPV viral-like particles (HMPV-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two HMPV viruses of either lineage A or B. These VLPs functionally display F and G HMPV surface glycoproteins. When injected in mice, HMPV-VLPs induce strong humoral immune response against both homologous and heterologous strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent infection of the lungs with both homologous and heterologous viruses. Upon challenge, viral titers in the lungs of immunized animals were significantly reduced as compared to those of control animals. In conclusion, a HMPV-VLP vaccine that induces cross-protective immunity in mice is a promising approach to prevent HMPV infections.     

Effect of vaccine delivery system on the induction of HPV16L1-specific humoral and cell-mediated immune responses in immunized rhesus macaques

There have been numerous studies to assess the immunogenicity of candidate therapeutic and prophylactic vaccines for human papillomavirus (HPV), but few of them have directly compared different vaccines in an immunologically relevant animal system. In the present study, several vaccine delivery systems (VLPs, chimeric VLPs, plasmid DNA, and a replication incompetent adenoviral vector) expressing HPV16L1 were evaluated for their ability to induce HPV16L1 VLP-specific humoral immune responses, including neutralizing antibodies, and cell-mediated immune responses in rhesus macaques. Monkeys immunized with HPV16L1 VLPs mounted a potent humoral response with strongly neutralizing antibodies and a strong L1-specific Th2 response as measured by IL-4 production by CD4+ T cells. Monkeys immunized with plasmid DNA or an adenoviral vector expressing HPV16L1 showed strong Th1/Tc1 responses as measured by IFN-gamma production by CD4+ and/or CD8+ T cells and potent humoral responses, but only weakly neutralizing antibodies. These data demonstrate that the nature of the immune response against HPV16L1 is dramatically different when it is introduced via different delivery systems. Additionally, these findings support the notion that an HPV16L1 VLP-based vaccine will induce the strongly neutralizing antibodies necessary for effective prophylaxis.     

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells

Recent phase IIb/III trials of a tetravalent live attenuated vaccine candidate revealed a need for improvement in the stimulation of protective immunity against diseases caused by dengue type 2 virus (DENV-2). Our attempts to develop particulate antigens for possibly supplementing live attenuated virus preparation involve generation and purification of recombinant DENV-2 virus-like particles (VLPs) derived from stably (prM+E)-expressing mosquito cells. Two VLP preparations generated with either negligible or enhanced prM cleavage exhibited different proportions of spherical particles and tubular particles of variable lengths. In BALB/c mice, VLPs were moderately immunogenic, requiring adjuvants for the induction of strong virus neutralizing antibody responses. VLPs with enhanced prM cleavage induced higher levels of neutralizing antibody than those without, but the stimulatory activity of both VLPs was similar in the presence of adjuvants. Comparison of EDIII-binding antibodies in mice following two adjuvanted doses of these VLPs revealed subtle differences in the stimulation of anti-EDIII binding antibodies. In cynomolgus macaques, VLPs with enhanced prM cleavage augmented strongly neutralizing antibody and EDIII-binding antibody responses in live attenuated virus-primed recipients, suggesting that these DENV-2 VLPs may be useful as the boosting antigen in prime-boost immunization. As the levels of neutralizing antibody induced in macaques with the prime-boost immunization were comparable to those infected with wild type virus, this virus-prime VLP-boost regimen may provide an immunization platform in which a need for robust neutralizing antibody response in the protection against DENV-2-associated illnesses could be tested.     

Recombinant heat shock protein 65 carrying hepatitis B core antigen induces HBcAg-specific CTL response

Many studies have provided evidence that heat shock protein 65 (Hsp65) can elicit potent specific cellular adaptive immune responses (e.g. CD8(+) cytotoxic T-cell effectors or classic CTLs) based on their ability to chaperone antigenic peptides. Hsp65 is thus an effective carrier for heterologous peptide epitopes for therapeutic vaccines against cancer or chronic infectious diseases. The core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, and functions as both a T-cell-dependent and a T-cell-independent antigen. Therefore, HBcAg may be a promising candidate target for therapeutic vaccine control of chronic HBV infection. Here, a chimeric protein, Hsp65Bc, was created by fusing the HBcAg sequence to the carboxyl terminus of the Hsp65 sequence in E. coli. Analysis of its antigenicity and immunogenicity revealed that HBc epitopes are surface accessible. Hsp65Bc induced moderate anti-HBc immune responses as well as a strong specific T-cell response in BALB/c mice. These results indicate that Hsp65Bc may have potential as a vaccine for treatment of HBV chronic infection.     

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.     

Chimeric calicivirus-like particles elicit specific immune responses in pigs

Virus-like particles (VLPs) have received considerable attention due to their potential application in veterinary vaccines and, in particular, VLPs from rabbit haemorrhagic disease virus (RHDV) have successfully shown to be good platforms for inducing immune responses against an inserted foreign epitope in mice. The aim of this study was to assess the immunogenicity of chimeric RHDV-VLPs as vaccine vectors in pigs. For this purpose, we have generated chimeric VLPs containing a well-known T epitope of 3A protein of foot-and-mouth disease virus (FMDV). Firstly, RHDV-VLPs were able to activate immature porcine bone marrow-derived dendritic cells (poBMDCs) in vitro. Secondly, pigs were inoculated twice in a two-week interval with chimeric RHDV-VLPs at different doses intranasally or intramuscularly. One intramuscularly treated group was also inoculated with adjuvant Montanide™ ISA 206 at the same time. Specific IgG and IgA antibodies against RHDV-VLPs were induced and such levels were higher in the adjuvanted group compared with other groups. Interestingly, anti-RHDV-VLP IgA responses were higher in groups inoculated intramuscularly than those that received the VLPs intranasally. Two weeks after the last immunisation, specific IFN-γ-secreting cells against 3A epitope and against RHDV-VLPs were detected in PBMCs by ELISPOT. The adjuvanted group exhibited the highest IFN-γ-secreting cell numbers and lymphoproliferative specific T cell responses against 3A epitope and RHDV-VLP. This is the first immunological report on the potential use of chimeric RHDV-VLPs as antigen carriers in pigs.     

Effect of HIV-1 envelope cytoplasmic tail on adenovirus primed virus encoded virus-like particle immunizations

The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env. Truncated Env overexpressed VRC01 and 17b binding antigen on the surface of transduced cells while the full length Env vaccine presented more and similar amounts of antigen binding to the trimer conformation sensitive antibodies PGT151 and PGT145, respectively. The adenoviral vectors were used to prime Balb/c mice followed by sequential boosting with chimpanzee type 63, and chimpanzee type 3 adenoviral vectors encoding SIVmac239 Gag and full length consensus Env. Both vaccine regimens induced increasing titers of binding antibody responses after each immunization, and significant differences in immune responses between the two groups were observed after the final immunization. Full length Env priming skewed antibody responses towards gp41, while truncated Env priming induced responses primarily targeting gp120 containing and derived antigens. Importantly, no differences in neutralizing antibody responses were found between the different priming regimens as both induced high titered tier 1 neutralizing antibodies, but no tier 2 antibodies, possibly reflecting the similar presentation of trimer specific antibody epitopes. The described vaccine regimens provide insight into the effects of the HIV-1 Env cytoplasmic tail on epitope presentation and subsequent immune responses, which is relevant for the interpretation of current clinical trials that are using truncated Env as an immunogen. The regimens described here provide similar neutralization titers, and thus are useful for investigating the importance of specificity in non-neutralizing antibody mediated protection against viral challenge.     

Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents.     

A hantavirus nucleocapsid protein segment exposed on hepatitis B virus core particles is highly immunogenic in mice when applied without adjuvants or in the presence of pre-existing anti-core antibodies

Hepatitis B virus (HBV) core particles carrying the amino-terminal 120 amino acids (aa) of the nucleocapsid (N) protein of the hantaviruses Dobrava, Hantaan or Puumala have been demonstrated to be highly immunogenic in mice when complexed with adjuvants. Here we demonstrate that even without adjuvant, these chimeric particles induced high-titered, and strongly cross-reactive N-specific antibody responses in BALB/c and C57BL/6 mice. The induced N-specific antibodies represented all IgG subclasses. Pre-existing core-specific antibodies did not abrogate the induction of an N-specific immune response by a hantavirus N insert presented on core particles. Therefore, chimeric core particles should represent promising vaccine candidates even for anti-core positive humans.     

Hepatitis C virus envelope glycoprotein immunization of rodents elicits cross-reactive neutralizing antibodies

Neutralizing antibody responses elicited during infection generally confer protection from infection. Hepatitis C virus (HCV) encodes two glycoproteins E1 and E2 that are essential for virus entry and are the major target for neutralizing antibodies. To assess whether both glycoproteins are required for the generation of a neutralizing antibody response, rodents were immunized with a series of glycoproteins comprising full length and truncated versions. Guinea pigs immunized with HCV-1 genotype 1a E1E2p7, E1E2 or E2 generated high titer anti-glycoprotein antibody responses that neutralized the infectivity of HCVpp and HCVcc expressing gps of the same genotype as the immunizing antigen. Less potent neutralization of viruses bearing the genotype 2 strain J6 gps was observed. In contrast, immunized mice demonstrated reduced anti-gp antibody responses, consistent with their minimal neutralizing activity. Immunization with E2 alone was sufficient to induce a high titer response that neutralized HCV pseudoparticles (HCVpp) bearing diverse glycoproteins and cell culture grown HCV (HCVcc). The neutralization titer was reduced 3-fold by the presence of lipoproteins in human sera. Cross-competition of the guinea pig anti-E1E2 immune sera with a panel of epitope mapped anti-E2 monoclonal antibodies for binding E2 identified a series of epitopes within the N-terminal domain that may be immunogenic in the immunized rodents. These data demonstrate that recombinant E2 and E1E2 can induce polyclonal antibody responses with cross-reactive neutralizing activity, supporting the future development of prophylactic and therapeutic vaccines.