Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are major causes of illness among children, the elderly, and the immunocompromised. No vaccine has been licensed for protection against either of these viruses. We tested the ability of two Venezuelan equine encephalitis virus-based viral replicon particle (VEE-VRP) vaccines that express the hRSV or hMPV fusion (F) protein to confer protection against hRSV or hMPV in African green monkeys. Animals immunized with VEE-VRP vaccines developed RSV or MPV F-specific antibodies and serum neutralizing activity. Compared to control animals, immunized animals were better able to control viral load in the respiratory mucosa following challenge and had lower levels of viral genome in nasopharyngeal and bronchoalveolar lavage fluids. The high level of immunogenicity and protective efficacy induced by these vaccine candidates in nonhuman primates suggest that they hold promise for further development.     

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 microg of pNJEME by intramuscular (i.m.) injection, and 3 microg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 microg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 microg of pNJEME or by gene gun administration with 3 microg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.     

Replication deficient human adenovirus vector serotype 19a/64: Immunogenicity in mice and female cynomolgus macaques

The human adenovirus type 19a/64 (hAd19a) is a rare serotype in the human population that transduces human dendritic cells (DCs) and human muscle cells more efficiently than the well-characterized human adenovirus type 5 (hAd5).To further characterize the potential of this vector as a vaccine we designed replication deficient hAd19a, hAd5 and MVA vectors expressing a papillomavirus (PV) antigen fused to the human MHC class II associated invariant chain T cell adjuvant (hIi) and investigated their immunogenicity in vivo in mice and cynomolgus macaques.We initially showed that the hIi encoded in the hAd5 enhanced PV specific CD8+ T cell responses in mice. The T cell responses induced after hAd19a vaccination was similar to those induced by hAd5 vaccination. The hAd19a induced responses were not reduced in presence of preexisting Ad5 immunity in mice. In macaques both vaccines were equally potent at inducing CD8+ T cells after MVA boost, while the level of CD4+ T cell responses were found to be broader in hAd19a primed animals.These data demonstrate the potential of hAd19a as an alternative vector to hAd5 to elicit potent T cell responses to PV.     

Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens

West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA–WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA–WNV vaccines in other preclinical models and use them as candidate vaccine in humans.     

Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines

Since intradermal delivery of DNA vaccines via tattoo device is an efficient strategy to induce antigen-specific immune responses, we evaluated this route of application for adenoviral vector vaccines in mice. Although expression levels were comparable after i.d. injection and i.d. tattoo immunization of adenoviral vectors, the tattoo application confined antigen expression to the upper layers of the dermis. Both delivery approaches resulted in strong CD8+ T-cell and humoral immune responses to three different antigens and conferred protection against mucosal challenge with respiratory syncytial virus. However, in contrast to results obtained with DNA vaccines, intradermal tattoo immunization did not provide any obvious advantage in comparison to simple intradermal injection of the adenoviral vector vaccines.     

Immunogenicity of two formulations of oral cholera vaccines in Thai volunteers

A formulation of oral vaccine consisting of Vibrio cholerae lipopolysaccharides (LPS), cell-bound haemagglutinin (CHA) and procholeragenoid (P), namely vaccine A, was compared with another formulation, vaccine B, prepared from killed whole vibrios plus procholeragenoid on their immunogenicity and reactogenicity in Thai male volunteers. Volunteers were randomly allocated into three groups. The first two groups received orally three doses of vaccines A and B, respectively at 14-day intervals. Volunteers in group 3 were controls and received orally 100 ml 5% (w/v) NaHCO3 also at 14-day intervals. Serum samples were collected from all volunteers before each immunization. Intestinal lavage was performed 3 to 7 days before the first dose of vaccine or placebo and 7, 21 and 45 days after the last dose. Serum vibriocidal antibodies were determined and class-specific, antigen-specific antibodies of all serum and lavage samples were assessed by indirect enzyme-linked immunosorbent assay (ELISA) using purified LPS, CHA and cholera toxin (CT) as antigens. Diarrhoea occurred in 10 and 40% of the vaccinees ingesting the vaccines A and B, respectively. The immunogenicity of the vaccine B in terms of seroconversion for vibriocidal antibodies and anti-LPS was higher than the vaccine A. Both vaccines had equal immunogenicity concerning serum anti-CT, while the vaccine A was slightly better than the vaccine B on serum anti-CHA response. The immunogenicity of the two vaccines in evoking intestinal responses was different from the systemic one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection

Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is an important cause of acute respiratory tract disease. In the 1960s, vaccination with formalin-inactivated paramyxovirus preparations--respiratory syncytial virus (RSV) and measles virus (MV)--resulted in predisposition for enhanced disease upon natural infection. We have produced a formalin-inactivated hMPV preparation (FI-hMPV), which was used to immunize young cynomolgus macaques. Six days after challenge FI-hMPV-primed monkeys had developed eosinophilic bronchitis and bronchiolitis, indicative of a hypersensitivity response. This study indicates that formalin-inactivated hMPV vaccines have the same propensity to predispose for immune-mediated disease as inactivated RSV and MV vaccines.     

Immunogenicity and efficacy of recombinant subunit vaccines against phocid herpesvirus type 1

Phocid herpesvirus type 1 (PhHV-1) is an alpha-herpesvirus that causes significant morbidity and mortality among young and immunocompromised harbour seals (Phoca vitulina) and therefore represents a major problem for seal rehabilitation centres. Consequently, there is a need for a safe and effective PhHV-1 vaccine. We tested an ISCOM-based recombinant PhHV-1 gB vaccine alone (gB) or with the addition of recombinant PhHV-1 gD (gBD) for (i). immunogenicity and protective efficacy against feline herpesvirus (FHV) infection in cats and (ii). their immunogenicity in seals. The FHV-cat model was chosen based on the close antigenic relationship between PhHV-1 and FHV. Upon challenge, all vaccinated (gB and gBD) cats excreted significantly less FHV (P<0.01) and gBD vaccinated cats showed less weight loss (P=0.05) than the mock-vaccinated cats. However, adding gD to the gB vaccine did not result in significantly better protection. Based on these data, immunogenicity studies in seals under rehabilitation were performed with the gB vaccine only. To this end, gB vaccine was tested at two different doses (20 or 40 microg). PhHV-1 specific antibody titres and in vitro proliferative T cell responses were measured in all seals upon vaccination. No differences were observed in antibody titres between seals vaccinated with either 20 or 40 microgB, but the higher gB concentration did result in higher specific proliferative T cell responses (P<0.01). Based on the close antigenic relationship between PhHV-1 and FHV, the safety and efficacy data in the FHV-cat model, and the immunogenicity data in the vaccinated seals, the gB based vaccine is expected to be safe and effective in protecting against PhHV-1 related disease in harbour seals.     

Immunogenicity and efficacy of two types of West Nile virus-like particles different in size and maturation as a second-generation vaccine candidate

Virus-like particles (VLPs) of flaviviruses generated from the prM and E genes are a promising vaccine candidate. We have established cell clones continuously releasing VLPs of West Nile virus (WNV) in serum-free conditions. Two types of VLPs were distinguished by sedimenting analyses in sucrose density gradients. Fast sedimenting VLPs (F-VLPs) were large (40–50 nm) and composed of the E and processed mature M proteins, whereas slowly sedimenting VLPs (S-VLPs) were small (20–30 nm) particles consisting of the E and immature prM proteins. F-VLPs induced higher neutralizing antibody and anti-WNV IgG titers than S-VLPs. Furthermore, IgG2a was dominant over IgG1 by immunization with F-VLPs as with whole virion-derived antigens. Mice vaccinated with a low dose (3 ng) of F-VLPs showed higher protective efficacy (83% survivals) against WNV infection than S-VLP-immune mice (17% survivals). These results indicate that F-VLPs more closely resemble the virions and take a better immunogenic form than S-VLPs as WNV vaccine candidates.     

Immunogenicity and protective efficacy of a psoralen-inactivated dengue-1 virus vaccine candidate in Aotus nancymaae monkeys

Psoralens are photoreactive compounds that cross-link pyrimidines after exposure to UVA radiation. In this experiment, we tested the protective efficacy of a psoralen-inactivated dengue vaccine candidate in non-human primates. Two groups of 7 Aotus nancymaae monkeys received either 10 ng per dose of inactivated DENV1 plus alum adjuvant or alum alone (controls). Doses were injected intradermally on days 0, 14, and 28. Monkeys then received a challenge inoculation of 1.1 × 10⁴ PFUs of WestPac 74 DENV-1 on day 132. At 62 days, only 1/7 vaccinated monkeys had detectable IgM, but IgG and neutralizing antibody remained detectable in 7/7. No IgM, IgG, or neutralizing antibody was detectable in control monkeys. DENV-1 viremia was detected after challenge in 3/7 vaccinated monkeys and 5/6 control monkeys (with one removed due to pregnancy) (p = 0.27), but days of viremia were reduced from 3.67 days/animal among controls to 0.71 days/animal among vaccinated monkeys (p = 0.051). Psoralen-inactivated DENV1 is immunogenic in Aotus nancymaae with a trend towards a reduction in days of viremia following experimental challenge.     

Protective efficacy of two new anti-caries DNA vaccines

As we know, the catalytic region of glucosyltransferases (GTFs) is a key region responsible for sucrose-dependent adherence of cariogenic bacteria to teeth. In this study, we evaluate the potential of the catalytic region to enhance the immunogenicity of the anti-caries DNA vaccine. We construct two new anti-caries DNA plasmids pGJGAC/VAX and pGJGA-5C/VAX by cloning different styles of the catalytic regions of GTFs into the previous plasmid pGJA-P/VAX. One is the 1.1 kb full length catalytic region of S. sobrinus GTF-I, the other is its catalytic core sequence which is conserved in the GTFs from mutans streptococci and plays a central role in the enzymatic activities of sucrose splitting and glucan synthesis. The results of caries protection experiment indicate that compared to pGJA-P/VAX, immunization with both new plasmids provides more effective protection against cariogenic bacteria, especially against S. sobrinus. The plasmid encoding full length catalytic region could provide more effective protection against cariogenic bacteria than that encoding catalytic core conserved sequence even though the differences are not very dramatic.     

Immunogenicity and efficacy of codon optimized DNA vaccines encoding the F-protein of respiratory syncytial virus

Respiratory syncytial virus F-protein (RSV-F) is poorly expressed from DNA expression plasmids containing the wild type RSV-F open reading frame. By codon optimization, premature polyadenylation signals were deleted and a striking enhancement of RSV-F expression levels was achieved. Therefore, the immunogenicity and efficacy of wild type DNA vaccines were compared to codon optimized expression plasmids encoding full-length RSV-F or its ectodomain. Mice were immunized twice with the different DNA vaccines followed by an RSV challenge. Only codon optimized DNA vaccines and in particular the one encoding the ectodomain of RSV-F induced substantial antibody levels and reduced viral load 13-170-fold. Thus, codon optimization enhances the immunogenicity and efficacy of RSV encoding DNA vaccines.     

Immunogenicity and in vitro protective efficacy of a polyepitope Plasmodium falciparum candidate vaccine constructed by epitope shuffling

A polyepitope chimeric antigen incorporating multiple protective and conservative epitopes from multiple antigens of Plasmodium falciparum has been considered to be more effective in inducing multiple layers of immunity against malaria than a single stage- or single antigen-based vaccine. By modifying the molecular breeding approach to epitope shuffling, we have constructed a polyepitope chimeric gene that encodes 11 B-cell and T-cell proliferative epitope peptides derived from eight key antigens mostly in the blood stage of Plasmodium falciparum. A 35-kDa antigen encoded by this gene, named Malaria RCAg-1, was purified from an E. coli expression system. Immunization of rabbits and mice with the purified protein in the presence of Freund's adjuvant strongly generated long-lasting antibody responses that recognized the corresponding individual epitope peptide in this vaccine as well as blood stage parasites. CD4(+) T-cell responses were also elicited as shown by the enhancement of T-cell proliferation, IFN-gamma and IL-4 level. In vitro assay of protection revealed that vaccine-elicited antibodies could efficiently inhibit the growth of blood-stage parasites. Additionally, the chimeric antigen was recognized by human serum specimens from malaria patients and individuals living in the endemic area. Our studies indicate the potential of M.RCAg-1 recombinant protein as malaria candidate vaccines as well as the rationale of the epitope shuffling technology applied in designing malaria vaccines.     

Inactivated whole influenza virus particle vaccines induce neutralizing antibodies with an increase in immunoglobulin gene subclones of B-lymphocytes in cynomolgus macaques

The All-Japan Influenza Vaccine Study Group has been developing a more effective vaccine than the current split vaccines for seasonal influenza virus infection. In the present study, the efficacy of formalin- and/or β-propiolactone-inactivated whole virus particle vaccines for seasonal influenza was compared to that of the current ether-treated split vaccines in a nonhuman primate model. The monovalent whole virus particle vaccines or split vaccines of influenza A virus (H1N1) and influenza B virus (Victoria lineage) were injected subcutaneously into naïve cynomolgus macaques twice. The whole virus particle vaccines induced higher titers of neutralizing antibodies against H1N1 influenza A virus and influenza B virus in the plasma of macaques than did the split vaccines. At challenge with H1N1 influenza A virus or influenza B virus, the virus titers in nasal swabs and the increases in body temperatures were lower in the macaques immunized with the whole virus particle vaccine than in those immunized with the split vaccine. Repertoire analyses of immunoglobulin heavy chain genes demonstrated that the number of B-lymphocyte subclones was increased in macaques after the 1st vaccination with the whole virus particle vaccine, but not with the split vaccine, indicating that the whole virus particle vaccine induced the activation of vaccine antigen-specific B-lymphocytes more vigorously than did the split vaccine at priming. Thus, the present findings suggest that the superior antibody induction ability of the whole virus particle vaccine as compared to the split vaccine is attributable to its stimulatory properties on the subclonal differentiation of antigen-specific B-lymphocytes.     

Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs

Three short fragments of recombinant subunit Pasteurella multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. PMT-specific antibody secreting cells and evidence of cellular immunity were detected in rsPMT-immunized pigs following authentic PMT challenge or homologous antigen booster. Piglets immunized with rsPMT fragments containing either the N-terminal or the C-terminal portions of PMT developed high titers of neutralizing antibodies. Pregnant sows immunized with rsPMT had higher levels of maternal antibodies in their colostrum than did those immunized with a conventional PAR-toxoid vaccine. Offspring from rsPMT vaccinated sows had better survival after challenge with a five-fold lethal dose of authentic PMT and had better growth performance after challenge with a sublethal dose of toxin. Our findings indicate these non-toxic rsPMT proteins are attractive candidates for development of a subunit vaccine against PAR in pigs.     

Comparative efficacy of two next-generation Rift Valley fever vaccines

Rift Valley fever virus (RVFV) is a re-emerging zoonotic bunyavirus of the genus Phlebovirus. A natural isolate containing a large attenuating deletion in the small (S) genome segment previously yielded a highly effective vaccine virus, named Clone 13. The deletion in the S segment abrogates expression of the NSs protein, which is the major virulence factor of the virus. To develop a vaccine of even higher safety, a virus named R566 was created by natural laboratory reassortment. The R566 virus combines the S segment of the Clone 13 virus with additional attenuating mutations on the other two genome segments M and L, derived from the previously created MP-12 vaccine virus. To achieve the same objective, a nonspreading RVFV (NSR-Gn) was created by reverse-genetics, which not only lacks the NSs gene but also the complete M genome segment. We have now compared the vaccine efficacies of these two next-generation vaccines and included the Clone 13 vaccine as a control for optimal efficacy. Groups of eight lambs were vaccinated once and challenged three weeks later. All mock-vaccinated lambs developed high fever and viremia and three lambs did not survive the infection. As expected, lambs vaccinated with Clone 13 were protected from viremia and clinical signs. Two lambs vaccinated with R566 developed mild fever after challenge infection, which was associated with low levels of viral RNA in the blood, whereas vaccination with the NSR-Gn vaccine completely prevented viremia and clinical signs.     

Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c)

In this study we have evaluated the vaccine potential of a Mycobacterium tuberculosis antigen of the PPE protein family, namely PPE44 (Rv2770c). PPE44-specific immune responses could be detected in mice acutely, chronically and latently infected with M. tuberculosis. Vaccination of mice with a plasmid DNA vaccine coding for PPE44 or recombinant PPE44 protein formulated in adjuvant generated strong cellular and humoral immune responses; immunodominant T cell epitopes were identified. Most importantly, vaccination of mice with both subunit vaccines followed by an intratracheal challenge with M. tuberculosis resulted in a protective efficacy comparable to the one afforded by BCG. Taken together these results indicate that PPE44 of M. tuberculosis is a protective antigen that could be included in novel subunit TB vaccines and that warrants further analysis.     

Immunogenicity, efficacy, safety and effectiveness of pneumococcal conjugate vaccines (1998-2006)

In this paper we present an overview of the literature on efficacy and safety trials of the various pneumococcal conjugate vaccines on the market (PCV7) and in development (PCV9, PCV11 and allegedly PCV10 and PCV13), as well as of observations from post-licensure studies. Seven- (PCV7) and nine-valent PCV (PCV9) are reported to be sufficiently immunogenic after administration of a 3+1 schedule in infants in various RCTs. PncOMPC (PCV7 with a protein of N. meningitidis as a carrier) is less immunogenic, though this may have no repercussions for the protective efficacy against clinical disease. PCV7 is 82-97% efficacious against vaccine serotype (VT) IPD, 90% efficacious against (clinically diagnosed) pneumococcal pneumonia, and, like the 11-valent PCV, 57% efficacious against VT acute otitis media. Naturally, it would be of paramount public health interest if the same levels of efficacy and effectiveness could be achieved with fewer doses. Trials studying 2+1 vaccination schedules for PCV7 and PCV9 generally show that the percentage of infants achieving the protective cut-off set by the World Health Organization (WHO) 1 month after the last priming dose, is comparable to that found at the same time point in studies administering 3+1 schedules. PCVs are generally very well tolerated and safe, also when co-administered with other childhood vaccines. As more and more countries are using these vaccines routinely, post marketing surveillance studies will further establish the safety profile of PCVs.     

Porcine rotavirus strain Gottfried-based human rotavirus candidate vaccines: construction and characterization

Rotavirus gastroenteritis remains the leading cause of severe diarrheal disease in infants and young children worldwide, and thus, a safe and effective rotavirus vaccine is urgently needed in both developing and developed countries. Various candidate rotavirus vaccines that were developed by us and others have been or are being evaluated in different populations in various parts of the world. We have recently confirmed that a porcine rotavirus Gottfried strain bears a P (VP4) serotype (P2B[6]) closely related to human rotavirus P serotype 2A[6] which is of epidemiologic importance in some regions of the world. Based on the modified Jennerian approach to immunization, we have constructed 11 Gottfried-based single VP7 or VP4 gene substitution reassortant vaccine candidates which could provide: (i) an attenuation phenotype of a porcine rotavirus in humans; and (ii) antigenic coverage for G serotypes 1-6 and 8-10 and P serotype 1A[8], 1B[4] and 2A[6]. In addition, following immunization of guinea pigs with Gottfried VP4, we found low but consistent levels of neutralizing antibodies to VP4 with P1A[8] or P1B[4] specificity, both of which are of global epidemiologic importance. Thus, porcine-based VP7 reassortant rotavirus vaccines may provide an advantage over rhesus- or bovine-based VP7 reassortant vaccines since the VP4s of the latter vaccines do not evoke antibodies capable of neutralizing the viruses bearing P1A[8], P1B[4] or P2A[6] VP4.