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.     

Effects of targeted fusion anti-caries DNA vaccine pGJA-P/VAX in rats with caries

Our previously prophylactic studies have proved that the anti-caries DNA vaccine pGJA-P/VAX could generate effective immune response by intramuscular (i.m.) and intranasal (i.n.) administration in rats without caries. In the present, we determine whether it also could produce efficacy in rats with caries. By immunized with pGJA-P/VAX, rats were elicited both significantly higher anti-Streptococcus mutans serum IgG and salivary SIgA responses, compared to those with pVAX1. Correspondingly, rats immunized with pGJA-P/VAX via i.n. displayed significantly fewer enamel, dentinal lesions compared to those with pVAX1 via i.n. However, there was no significant difference in dental caries lesions between pGJA-P/VAX (i.m.) and pVAX1 (i.m.). These findings suggest that DNA vaccination via i.n., with bupivacaine delivery system, could be a promising alternatives for slowing down caries development in rat models.     

Immunogenicity of a hexavalent combination vaccine in rhesus monkeys

Preclinical immunogenicity studies were conducted in rhesus monkeys to determine whether there is immune interference in the response to one or more components of a hexavalent vaccine (Hexavac) that contains antigens from Haemophilus influenzae (Hib), hepatitis B (HB), diphtheria (D), tetanus (T), acellular pertussis (aP) and inactivated polio virus (IPV). Antibody responses were measured following co-administration of the components at three separate anatomical sites or administration as a hexavalent combination in a single site. After three injections of the hexavalent vaccine, the peak antibody responses to each component of the vaccine were >100-fold above pre-immune titers and persisted at levels >10-fold above pre-immune titers at approximately 1 year. Immune interference was observed in the peak response to HB, D and pertussis toxin, but was not seen at later time points. The results indicate that the rhesus monkey model may be useful for pre-clinical evaluation of combination vaccines.     

Mucosal and systemic immunization with targeted fusion anti-caries DNA plasmid in young rats

Early life vaccination is necessary to protect young children from dental caries. Our group had previously reported that a plasmid DNA vaccine pGJA-P/VAX against the glucosyltransferase (GTF) enzyme and cell surface antigen AgI/II (PAc) of Streptococcus mutans (S. mutans) elicited a specific and protective immunity in adult experimental animal models. In this report, early life immunization with the same plasmid was studied following intranasal (i.n.) and intramuscular (i.m.) delivery in murine models. The potential of inducing mucosal and systemic immune responses to special antigens was measured by ELISA. In addition, cytokine production and protection effectiveness against dental caries formation were also investigated. In the i.n. route, rats were primed when they were 5 days old, and boosted after 10 and 20 days with either plasmid pGJA-P/VAX–bupivacaine complexes, or pGJA-P/VAX alone, or empty vector. The pGJA-P/VAX–bupivacaine combination was able to mount the immune responses characterized by increased antibody levels of specific salivary IgA and serum IgG, preferential IFN-γ production and significant reduction in the dental caries lesions. In the i.m. route, rats were vaccinated with either pGJA-P/VAX alone or empty vector with the same immunization schedule as the i.n. route. Plasmid pGJA-P/VAX alone induced a significant increase in the serum IgG and IFN-γ production. However, it was not effective in eliciting specific salivary IgA and in decreasing the dental caries formation. All these findings indicate the feasibility of immunity with a targeted fusion DNA vaccine to a young immune system.     

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine

BACKGROUND: In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys. METHODS: The cultures of SARS coronavirus (SARS-CoV) BJ-01 strain infected Vero cells were inactivated with beta-propiolactone. Sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated SARS vaccine. The purified SARS vaccine was analyzed with electron microscope, HPLC and Western blotting. We immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. Antibody titers were measured by plaque reduction neutralization test. The vaccinated monkeys were challenged with SARS-CoV BJ-01 strain to observe protective efficacy. Additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated SARS vaccine (0.5, 1 and 2mug/time/monkey) on days 0 and 7, and the monkeys were challenged with SARS-CoV GZ-01 strain. We assessed the safety of the SARS vaccine and observed whether the antibody dependent enhancement (ADE) occurred under low levels of neutralizing antibody in rhesus. FINDINGS: The purity of SARS vaccine was 97.6% by HPLC identification and reacted with convalescent sera of SARS patients. The purified SARS vaccine induced high levels of neutralizing antibodies and prevented the replication of SARS-CoV in monkeys. Under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with SARS-CoV. In this preliminary animal trial, no side effects were detected when monkeys were immunized with purified SARS vaccine either at normal or large doses. INTERPRETATION: The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.     

Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis

Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-γ and IL-2 response against kinesin (3012 ± 102 and 367.5 ± 8.92 pg/ml) and ESAT-6 (1334 ± 46.5 and 245.1 ± 7.72 pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-γ and IL-2) against individual construct was lower (kinesin motor domain: 1788 ± 36.48 and 341.8 ± 9.801 pg/ml and ESAT-6: 867.0 ± 47.23 and 170.8 ± 4.578 pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-γ production against chimera vaccination is statistically significant (p < 0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.     

Immunogenicity of an HPV-16 L2 DNA vaccine

The ability to elicit cross-neutralizing antibodies makes human papillomavirus (HPV) L2 capsid protein a possible HPV vaccine. We examined and compared the humoral response of mice immunized with a HPV-16 L2 DNA vaccine or with HPV-16 L2 protein. The L2 DNA vaccine elicited a non-neutralizing antibody response unlike the L2 protein. L2 DNA vaccination suppressed the growth of L2-expressing C3 tumor cells, which is a T cell mediated effect, demonstrating that the lack of non-neutralizing antibody induction by L2 DNA was not caused by lack of T cell immunogenicity of the construct.     

Immunogenicity of a heat-treated recombinant DNA hepatitis B vaccine

The effect of heating on the reactogenicity and the immunogenicity of a recombinant DNA hepatitis B vaccine was examined. Treatment of this vaccine for one week at 37 degrees C did not significantly alter these properties when compared to vaccine stored at 4 degrees C. These results have important implications in countries where facilities for the transport and handling of vaccines are often inadequate.     

Immunogenicity and protective efficacy of a tuberculosis DNA vaccine expressing a fusion protein of Ag85B-Esat6-HspX in mice

Tuberculosis remains a major infectious disease worldwide due to the low efficacy of available vaccine of the Mycobacterium bovis Bacillus Calmette-Guérin (BCG). DNA vaccines are especially promising candidates; however, the efficacy of DNA vaccine expressing single antigen of Mycobacterium tuberculosis (MTb) is limited. In this study, a plasmid DNA vaccine, pAEH, was constructed and designed to express a fusion protein of the Ag85B, Esat6, and HspX of MTb. Its immunogenicity and protective efficacy as well as therapeutic effect were assessed in a mouse model of tuberculosis. Vaccination with the pAEH significantly increased the frequency of peripheral blood CD4(+) and CD8(+) T cells, but not γδT cells, similar to that of vaccination with the BCG, and induced significantly higher levels of HspX-specific T cell proliferation, as compared with vaccination with BCG or the pHspX. Furthermore, vaccination with the pAEH increased the frequency of Ag85B, Esat6 and HspX-specific IFNγ-secreting T cells, accompanied by significantly higher levels of IFN-γ and IL-2 production ex vivo, as compared with that of the BCG or pHspX-vaccinated mice. Apparently, vaccination with the pAEH induced potent Th1 responses in mice. More importantly, vaccination with the pAEH inhibited the replication of virulent MTb in the lungs and spleens, even after MTb infection, and related lung inflammation in mice. Potentially, the newly developed pAEH vaccine may be used for the prevention and therapeutic intervention of MTb infection.     

Immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue DNA vaccine

A prototype dengue-1 DNA vaccine was shown to be safe and immunogenic in a previous Phase 1 clinical trial. Anti-dengue-1 neutralizing antibody responses were detectable only in the group of volunteers receiving the high dose of nonadjuvanted vaccine and the antibody titers were low. Vaxfectin^®, a lipid-based adjuvant, enhances the immunogenicity of DNA vaccines. We conducted a nonhuman primate study to evaluate the effect of Vaxfectin^® on the immunogenicity of a tetravalent dengue DNA vaccine. Animals were immunized on days 0, 28 and 84, with each immunization consisting of 3 mg of Vaxfectin^®-adjuvanted tetravalent dengue DNA vaccine. The use of Vaxfectin^® resulted in a significant increase in anti-dengue neutralizing antibody responses against dengue-1, -3 and -4. There was little to no effect on T cell responses as measured by interferon gamma ELISPOT assay. Animals immunized with the Vaxfectin^®-formulated tetravalent DNA vaccine showed significant protection against live dengue-2 virus challenge compared to control animals (0.75 mean days of viremia vs 3.3 days). Animals vaccinated with nonadjuvanted DNA had a mean 2.0 days of viremia. These results support further evaluation of the Vaxfectin^®-adjuvanted tetravalent dengue DNA vaccine in a Phase 1 clinical trial.     

Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits and monkeys

Bacillus anthracis, the causative agent of anthrax, is recognized as one of the most serious bioterrorism threats. The current human vaccines are based on the protective antigen component of the anthrax toxins. Concern about possible vaccine resistant strains and reliance on a single antigen has prompted the search for additional immunogens. Bacterial capsules, as surface-expressed virulence factors, are well-established components of several licensed vaccines. In a previous study we showed that an anthrax vaccine consisting of the B. anthracis poly-γ-d-glutamic acid capsule covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B protected mice against parenteral B. anthracis challenge. Here we tested this vaccine in rabbits and monkeys against an aerosol spore challenge. The vaccine induced anti-capsule antibody responses in both species, measured by ELISA and a macrophage opsono-adherence assay. While rabbits were not protected against a high aerosol challenge dose, significant protection was observed in monkeys receiving the capsule conjugate vaccine. The results confirm that the capsule is a protective immunogen against anthrax, being the first non-toxin antigen shown to be efficacious in monkeys and suggest that addition of capsule may broaden and enhance the protection afforded by protective antigen-based vaccines.     

Immunogenicity of a multicomponent DNA vaccine against visceral leishmaniasis in dogs

Vaccination of dogs, the domestic reservoir of Leishmania chagasi, could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-gamma (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific type 1 cellular immune response, which was able to restrict parasite growth.     

Immunogenicity, safety, and protective efficacy of an inactivated SARS-associated coronavirus vaccine in rhesus monkeys

An inactivated vaccine for severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) was evaluated in rhesus monkeys. The monkeys were inoculated intramuscularly (i.m.) with 0.5, 5, 50, or 5000 microg of vaccine, or PBS as control, and boosted on day 7. After 3 weeks, they were challenged with the NS-1 strain of SARS-CoV. The humoral and mucosal immune responses, clinical signs, chemical indices and viremia were monitored following the immunization and challenge. The control animals who received PBS developed atypical SAR-CoV infection after viral challenge, according to clinical, virological and pathological findings. No systematic side effects were observed in vaccinated animals post-immunization, even in at the high dose of 5000 microg. The 50 microg dosage of vaccine elicited SARS-CoV specific immune responses against viral infection as compared to the partial immunity elicited by 0.5 and 5 microg doses. The results show that this inactivated vaccine can induce effective concomitant humoral and mucosal immunity against SARS-CoV infection, is safe in monkeys, and the vaccine maybe a good candidate for clinical trials.     

Immunogenicity in mice and rabbits of DNA vaccines expressing woodchuck hepatitis virus antigens

The licensed vaccine against hepatitis B virus (HBV) is an effective means to prevent infection, but is not an effective therapeutic strategy to treat established chronic infections when used alone. In an animal model of chronic HBV infection (the woodchuck experimentally infected with woodchuck hepatitis virus (WHV)), the combination of conventional vaccine and potent antiviral drugs has shown promise as a potential therapeutic intervention. This approach might be improved further through the application of newer vaccine technologies. In the present study, we evaluated electroporation (EP)-based intramuscular (i.m.) delivery of a codon-optimized DNA vaccine for the WHV surface antigen (WHsAg) in mice and rabbits. In mice, this immunization procedure compared favorably to vaccination by i.m. injection of the DNA vaccine or i.m. administration of a recombinant WHsAg-alum vaccine, exhibiting characteristics expected to be beneficial for a therapeutic vaccine strategy. These included dose efficiency, consistency, vigorous induction of antibody responses to WHsAg, as well as a Th1 bias. Following scale-up to rabbits, a species that approximates the size of the woodchuck, the EP dosing regimen was markedly more effective than conventional i.m. injection of the DNA vaccine. Taken together, these results provide the foundation for studies of EP-based DNA immunization in the woodchuck in order to further assess its potential as an immunotherapeutic approach for treatment of chronic HBV infection in humans.     

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.     

Development of a recombinant tetravalent dengue virus vaccine: Immunogenicity and efficacy studies in mice and monkeys

Truncated recombinant dengue virus envelope protein subunits (80E) are efficiently expressed using the Drosophila Schneider-2 (S2) cell expression system. Binding of conformationally sensitive antibodies as well as X-ray crystal structural studies indicate that the recombinant 80E subunits are properly folded native-like proteins. Combining the 80E subunits from each of the four dengue serotypes with ISCOMATRIX^® adjuvant, an adjuvant selected from a set of adjuvants tested for maximal and long lasting immune responses, results in high titer virus neutralizing antibody responses. Immunization of mice with a mixture of all four 80E subunits and ISCOMATRIX^® adjuvant resulted in potent virus neutralizing antibody responses to each of the four serotypes. The responses to the components of the tetravalent mixture were equivalent to the responses to each of the subunits administered individually. In an effort to evaluate the potential protective efficacy of the Drosophila expressed 80E, the dengue serotype 2 (DEN2-80E) subunit was tested in both the mouse and monkey challenge models. In both models protection against viral challenge was achieved with low doses of antigen in the vaccine formulation. In non-human primates, low doses of the tetravalent formulation induced good virus neutralizing antibody titers to all four serotypes and protection against challenge with the two dengue virus serotypes tested. In contrast to previous reports, where subunit vaccine candidates have generally failed to induce potent, protective responses, native-like soluble 80E proteins expressed in the Drosophila S2 cells and administered with appropriate adjuvants are highly immunogenic and capable of eliciting protective responses in both mice and monkeys. These results support the development of a dengue virus tetravalent vaccine based on the four 80E subunits produced in the Drosophila S2 cell expression system.     

Immunogenicity of recombinant Modified Vaccinia Ankara following a single or multi-dose vaccine regimen in rhesus monkeys

Modified Vaccinia Ankara (MVA) is a replication-defective strain of vaccinia virus (VV) that is being investigated in humans as an alternative vaccine against smallpox. Understanding the parameters of a MVA vaccine regimen that can effectively enhance protective immunity will be important for clinical development. The present studies utilize cohorts of rhesus monkeys immunized with recombinant MVA (rMVA) or recombinant VV (rVV) vaccine vectors to investigate the magnitude, breadth, and durability of anti-VV immunity elicited by a single or multi-dose vaccine regimen. These data demonstrate that a single immunization with rMVA elicits weaker cellular and humoral immunity compared to a single inoculation with rVV. However, vaccine-elicited antibody responses, but not T cell responses, are significantly enhanced with repeated immunizations of rMVA. Importantly, only monkeys receiving up to four inoculations with rMVA generated neutralizing antibody (NAb) responses that were comparable in magnitude and durability to those elicited in monkeys receiving two inoculations with rVV. These data also show that the breadth of antibody responses against protein antigens associated with two antigenically distinct forms of infectious VV are similar in rMVA- and rVV-immunized monkeys. Together, these studies suggest that a multi-dose vaccine regimen utilizing up to four inoculations of MVA generates robust and durable antibody-mediated immunity comparable to that elicited by replication-competent VV.     

Immunogenicity of PCV24, an expanded pneumococcal conjugate vaccine,in adult monkeys and protection in mice

Invasive pneumococcal disease (IPD) is responsible for serious illnesses such as bacteremia, sepsis, meningitis, and pneumonia in young children, older adults, and persons with immunocompromising conditions and often leads to death. Although the most recent pneumococcal conjugate vaccines (PCVs) have been designed to target serotypes identified as the primary causative agents of IPD, the epidemiological landscape continues to change stressing the need to develop new PCVs. We have developed an investigational 24-valent PCV (PCV24) including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F all conjugated to CRM197 and evaluated this vaccine in adult monkeys. PCV24 was shown to be immunogenic and induced functional antibody for all vaccine serotypes. Of the serotypes common to PCV13 and V114 (PCV15), PCV24 had a similar immunogenic response with the exceptions of 23F which had higher IgG GMCs for PCV13 and V114, and 7F which had higher GMCs for PCV13. Functional antibody responses were similar for the serotypes in common between PCV24, PCV13 and V114 vaccines, with the exception of serotype 7F which was greater for PCV13. Overall, this study shows that PCV24 provided similar immunogenicity as the lower valent vaccines in adult monkeys with no apparent serotype interference. In addition, PCV24 also provided protection against pneumococcal infection in a mouse challenge model.     

Immunogenicity of adjuvanted plant-produced SARS-CoV-2 Beta spike VLP vaccine in New Zealand white rabbits

The outbreak of the SARS-CoV-2 global pandemic heightened the pace of vaccine development with various vaccines being approved for human use in a span of 24 months. The SARS-CoV-2 trimeric spike (S) surface glycoprotein, which mediates viral entry by binding to ACE2, is a key target for vaccines and therapeutic antibodies. Plant biopharming is recognized for its scalability, speed, versatility, and low production costs and is an increasingly promising molecular pharming vaccine platform for human health. We developed Nicotiana benthamiana-produced SARS-CoV-2 virus-like particle (VLP) vaccine candidates displaying the S-protein of the Beta (B.1.351) variant of concern (VOC), which triggered cross-reactive neutralising antibodies against Delta (B.1.617.2) and Omicron (B.1.1.529) VOCs. In this study, immunogenicity of the VLPs (5 µg per dose) adjuvanted with three independent adjuvants i.e. oil-in-water based adjuvants SEPIVAC SWE^TM (Seppic, France) and “AS IS” (Afrigen, South Africa) as well as a slow-release synthetic oligodeoxynucleotide (ODN) adjuvant designated NADA (Disease Control Africa, South Africa) were evaluated in New Zealand white rabbits and resulted in robust neutralising antibody responses after booster vaccination, ranging from 1:5341 to as high as 1:18204. Serum neutralising antibodies elicited by the Beta variant VLP vaccine also showed cross-neutralisation against the Delta and Omicron variants with neutralising titres ranging from 1:1702 and 1:971, respectively. Collectively, these data provide support for the development of a plant-produced VLP based candidate vaccine against SARS-CoV-2 based on circulating variants of concern.