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.     

Immunoprotection elicited by an enterovirus type 71 experimental inactivated vaccine in mice and rhesus monkeys

A number of commonly recognized public health issues are associated with EV71 infection, including the induction of severe cases of hand-foot-and-mouth disease (HFMD). Because of such issues, research and development of EV71 vaccine candidates is of growing importance. In the present study, an experimental EV71 inactivated vaccine was prepared, and its corresponding immunogenicity was analyzed. The immune responses and immunoprotective effect induced by the vaccine in mice and rhesus monkeys are described, and the two animal models are compared to evaluate the potential of assessing the inactivated vaccine's immunogenicity in these two species. In addition to assessing the vaccine's efficacy in mice, our data further elucidate the significance and value of assessing the immunogenicity and immunoprotection of vaccine candidates in rhesus monkeys by relying on a range of analyses, including pathological, etiological and lethal challenge analyses.     

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries. Currently, five vaccine candidates have been evaluated in clinical trials in China (three C4 candidates), Singapore (one B2 candidate), and Taiwan (one B4 candidate). Overall, the peak viral titers of these 5 vaccine candidates could only reach about 10⁷ TCID₅₀/mL. Moreover, genotypes of these 5 candidates are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia. We adapted a high-growth EV71 genotype B5 (HG-B5) virus after multiple passages and plaque selections in Vero cells and the HG-B5 virus could reach high titers (>10⁸ TCID₅₀/mL) in a microcarrier-based cell culture system. The viral particles were further purified and formulated with alum adjuvant. After two doses of intramuscular immunization in rabbits, the HG-B5 vaccine candidate could induce cross-reactive neutralizing antibodies against the three major EV71 genogroups. In conclusion, a high-growth EV71 virus was successfully adapted in Vero cells and could induce broad spectrum neutralizing antibody titers against three (A, B5, and C4) genotypes in rabbits.     

Dengue tetravalent DNA vaccine inducing neutralizing antibody and anamnestic responses to four serotypes in mice

We developed a dengue tetravalent DNA vaccine consisting of plasmids expressing premembrane and envelope genes of each of four serotypes of dengue viruses. BALB/c mice immunized twice with the tetravalent vaccine at a dose of 100 microg (25 microg for each serotype) using a needle-free jet injector developed neutralizing antibodies against all serotypes. There was no interference among the four components included in this combination vaccine. Tetravalent vaccine-immunized mice showed anamnestic neutralizing antibody responses following challenge with each dengue serotype: responses to challenges from serotypes different to those used for neutralization tests were also induced.     

Delineating the serotype-specific neutralizing antibody response to a live attenuated tetravalent dengue vaccine

The dengue virus (DENV) vaccines that are licensed or in clinical development consist of DENV serotype 1–4 tetravalent formulations given simultaneously and are not acquired sequentially like natural infections. It is unclear what effect this has on development of protective levels of immunity to all four serotypes. Serotype-specific neutralizing antibody (NAb) is considered the most relevant correlate of protection from dengue disease. Here we assessed levels of serotype-specific and cross-reactive NAb in immune sera from 10 subjects vaccinated with a live attenuated tetravalent DENV vaccine developed at the Walter Reed Army Institute of Research. The majority of subjects NAb responses to DENV-2 and DENV-4 were type-specific, while their NAb responses to DENV-1 and DENV-3 were primarily cross-reactive. Vaccine virus RNAemia has been most frequently detected for DENV-2 and DENV-4 in vaccinated subjects, strongly suggesting that replication is important for eliciting serotype-specific immunity.     

Focus and breadth of cellular immune responses elicited by a heterologous insert prime-boost vaccine regimen in rhesus monkeys

The global sequence diversity of HIV-1 presents a daunting challenge for vaccine development. We investigated whether a heterologous insert prime-boost regimen could expand global coverage by selectively boosting cellular immune responses to conserved epitopes. Rhesus monkeys were primed and boosted with recombinant adenovirus vectors expressing homologous or heterologous HIV-1 Gag sequences that were optimized to focus responses on highly conserved epitopes. We observed comparable responses directed to specific regions of the Gag protein in all experimental groups without evidence of improved coverage or expanded breadth in the heterologous insert group. These data suggest that antigen-independent factors contribute to the immunodominance patterns of vaccine-elicited cellular immune responses.     

Evaluation of Japanese encephalitis virus DNA vaccine candidates in rhesus monkeys [Macaca mulatta

We have previously described DNA vaccine candidates against Japanese encephalitis virus (JEV) that were immunogenic in mice. Present study was conducted to evaluate their immunogenicity in rhesus monkeys (Macaca mulatta) and compare it with the commercial mouse brain-derived, formalin-inactivated vaccine. Groups of four monkeys were immunized with either pMEa (expressing the anchored form of the envelope protein along with the pre-membrane protein of JEV) or pMEs (expressing the secretory form of the envelope protein along with pre-membrane protein of JEV) by intra-muscular (IM, using needle) or intra-dermal (ID, using gene gun) routes. Following primary immunization with 1mg plasmid DNA given IM, or 5 microg plasmid DNA given ID, the monkeys were boosted after 1 and 2 months with 0.5mg DNA given IM or 5 microg DNA given ID, and observed for a period of 6 months. After the second booster, most of the monkeys sero-converted and developed JEV neutralizing antibodies, albeit of low titer. Importantly however, following a sham challenge with the mouse brain-derived inactivated JEV vaccine given 6 months after immunization, the neutralizing antibody titers rose rapidly indicating a vigorous anamnestic response. Based on the JEV neutralizing antibody response following the vaccination and the extent of anamnestic response generated in the immunized monkeys, plasmid pMEa was superior to pMEs. This study indicates that the JEV candidate DNA vaccine is capable of generating protective levels of JEV neutralizing antibodies in rhesus monkeys and prime the immune system effectively against a subsequent exposure to JEV.     

Coxsackievirus A 16 infection does not interfere with the specific immune response induced by an enterovirus 71 inactivated vaccine in rhesus monkeys

Hand, foot and mouth disease is usually caused by enterovirus 71 (EV71) and coxsackievirus A 16 (CA16), which are members of the Picornaviridae family. In the present study, the characteristics of the immune response induced by an EV71 inactivated vaccine (made from human diploid cells) were explored in the presence of CA16 infection, based on the previously established neonatal rhesus monkey model. The typical clinical manifestations, including body temperature, viral viremia and virus shedding in the mouth, pharynx and feces, were characterized. A specific neutralizing antibody assay showed that the specific immune response induced by the EV71 inactivated vaccine was active against EV71 but not against CA16. No remarkable fluctuation in proinflammatory cytokine release was identified in the serum of immunized monkeys with EV71 vaccine and CA16 infections subsequently. The results showed that the specific immune response induced by the EV71 inactivated vaccine is effective against EV71 infection but is not affected by CA16 infection.     

Persistence of neutralizing antibody and its protective efficacy induced by a live attenuated tetravalent dengue vaccine,KD-382, in cynomolgus monkeys

An effective dengue vaccine should induce a long-lasting immune response against all four serotypes simultaneously with a minimum number of immunizations. Our live attenuated tetravalent dengue vaccine candidate, KD-382, was developed using a classical host range mutation strategy (no addition of artificial genetic modification). In our previous study, cynomolgus monkeys immunized with a single dose of KD-382 seroconverted to all four serotypes. However, it is important to determine if neutralizing antibodies (NAbs) induced by KD-382 can work as a long-lasting immune response to prevent dengue. In this study, a single dose of KD-382 induced a strong NAb response against all four serotypes in cynomolgus monkeys. We also confirmed that NAb titers against all four serotypes persist for at least five years, indicating its high potential as a dengue vaccine candidate. Next, we evaluated the effect of pre-existing dengue immunity on NAb responses induced by KD-382. We administered KD-382 to cynomolgus monkeys pre-administered one of the monovalent parental wild-type strains 60 days before vaccination. Regardless of the pre-immunized serotype, all the monkeys showed sufficient tetravalent NAb responses, which lasted for over two years. All the KD-382 vaccinated monkeys were then challenged with different parental wild-type viruses than that used for pre-administration; viral RNA in the serum was less than the lower limit of quantification, indicating complete protection against secondary heterologous dengue infection without any harmful disease enhancement. Consequently, KD-382 successfully induced a long-lasting and protective tetravalent NAb response in monkeys, suggesting that KD-382 is a promising vaccine candidate usable for both dengue seronegative and seropositive individuals.     

Immunogenicity and safety profile of a primary dose of bivalent oral polio vaccine given simultaneously with DTwP-Hb-Hib and inactivated poliovirus vaccine at the 4th visit in Indonesian infants

In this study, we aimed to evaluate the immunological protectivity of infants following four doses of bivalent oral polio vaccine (bOPV; Bio Farma), which were given simultaneously with DTwP-Hb-Hib (Pentabio®), along with one dose of inactivated poliovirus vaccine (IPV) at the fourth visit. A total of 143 newborn infants who fulfilled the inclusion criteria were enrolled and completed the study. Subjects received the first dose of bOPV at birth. On days 60, 90 and 120, bOPV was given simultaneously with Pentabio®. On day 120, one dose of IPV was also administered. Serum samples for serology analysis were collected before the first dose of bOPV (at day 0), before the second dose of bOPV (at day 60) and 30 days after the last dose of bOPV. In addition, the intensity, duration and relationship of each adverse event to the trial vaccines were assessed. Seroprotection rates after the fourth dose of bOPV were 100%, 91.6% and 99.3% for poliovirus P1, P2 and P3, respectively. Seroconversion rates after the fourth dose of bOPV were 100.0%, 93.3% and 100% for poliovirus P1, P2 and P3, respectively. There were no severe adverse events, and systemic reactions were generally mild during the 1–28 day post-vaccination period. Collectively, our findings indicate that bOPV given simultaneously with Pentabio® and one dose of IPV at the 4th visit was immunogenic and well tolerated.     

Oral administration of live Shigella vaccine candidates in rhesus monkeys show no evidence of competition for colonization and immunogenicity between different serotypes

Live oral monovalent Shigella flexneri 2a vaccine candidates as well as bivalent formulations with Shigella sonnei were evaluated in a rhesus monkey model for colonization and immunogenicity. Freshly harvested suspensions of S. flexneri 2a vaccine candidates WRSf2G12 and WRSf2G15 as well as S. sonnei vaccine candidate WRSs3 were nasogastrically administered to groups of rhesus monkeys, Macaca mulatta, either in a monovalent form or when combined with each other. The animals were monitored daily for physical well-being, stools were subjected to quantitative colony immunoblot assays for bacterial excretion and blood and stools were evaluated for humoral and mucosal immune responses. No clinical symptoms were noted in any group of animals and the vaccine candidates were excreted robustly for 48–72 h without significant changes in either the magnitude or duration of excretion when given as a monovalent or as bivalent mixtures. Similarly, immunological interferences were not apparent in the magnitude of humoral and mucosal immune responses observed toward Shigella-specific antigens when monkeys were fed monovalent or bivalent formulations. These results predict that a multivalent live oral vaccine of more than one serotype can have a favorable outcome for protection against shigellosis.     

Challenge studies in Rhesus monkeys immunized with candidate hepatitis E vaccines: DNA,DNA-prime-protein-boost and DNA-protein encapsulated in liposomes

Complete ORF2 gene (1983 bp) of hepatitis E virus (HEV) and the 450 bp region within ORF2 containing neutralizing epitope (NE) cloned in pVAX1 and corresponding proteins expressed in baculovirus and prokaryotic systems respectively were evaluated as vaccine candidates. Two doses of liposome encapsulated DNA plus corresponding protein with both ORF2 and NE regions (Lipo-ORF2-DP and Lipo-NE-DP) showed 100% seroconversion and comparable anti-HEV titres in Swiss albino mice. These vaccine candidates were further evaluated as DNA, DNA-prime-protein-boost (DPPB) and liposome formulations in Rhesus monkeys. Monkeys receiving ORF2/NE DNA seroconverted after fourth dose while those immunized employing ORF2-DPPB format seroconverted at 7 weeks post third dose. In view of the delayed weak antibody response, these monkeys were not challenged. Though Lipo-ORF2-DP was immunogenic, 2 of the 4 monkeys developed HEV infection following homologous virus challenge of 100 Monkey Infectious Dose₅₀. Both monkeys immunized with Lipo-NE-DP and 1 of the 2 monkeys immunized with NE-DPPB showed complete protection, the second monkey being protected from hepatitis with limited viral replication. Irrespective of the type of immunogen, all challenged monkeys were protected from hepatitis. The results document Lipo-NE-DP to be a promising vaccine candidate needing further evaluation.     

Unique cellular and humoral immunogenicity profiles generated by aerosol,intranasal, or parenteral vaccination in rhesus macaques

Respiratory mucosa immunization is capable of eliciting both local and distal mucosal immune responses; it is a potentially powerful yet largely unused modality for vaccination against respiratory diseases. Targeting the lower versus upper airways by aerosol delivery alters the immunogenicity profile of a vaccine, although the full extent of this impact is not well characterized. We set out to define the cellular and humoral response profiles elicited by immunization via intranasal, small aerosol droplets, and large aerosol droplets. We compared responses following adenovirus-vectored vaccination by these routes in macaques, either for the generation of primary immune responses or for the boosting of previously primed systemic responses. Aerosol delivery (4 or 10 μm diameter droplets, addressing lower or upper airways, respectively) generated the highest magnitude lung CD4 and CD8 T-cell responses, reaching 10–30% vaccine-specific levels in bronchoalveolar lavage cells. In contrast, intranasal delivery was less immunogenic with >10-fold lower peak lung T-cell responses. Systemic (blood) T-cell responses were only observed following 4 μm aerosol (and parenteral) immunization, while all delivery routes elicited similar humoral responses. These data demonstrate distinct immune response profiles with each respiratory tract vaccination modality and suggest that small droplet aerosol offers several immunological advantages over other respiratory routes.     

Development of a recombinant,chimeric tetravalent dengue vaccine candidate

Dengue is a significant threat to public health worldwide. Currently, there are no licensed vaccines available for dengue. Takeda Vaccines Inc. is developing a live, attenuated tetravalent dengue vaccine candidate (TDV) that consists of an attenuated DENV-2 strain (TDV-2) and three chimeric viruses containing the prM and E protein genes of DENV-1, -3 and -4 expressed in the context of the attenuated TDV-2 genome backbone (TDV-1, TDV-3, and TDV-4, respectively). TDV has been shown to be immunogenic and efficacious in nonclinical animal models. In interferon-receptor deficient mice, the vaccine induces humoral neutralizing antibody responses and cellular immune responses that are sufficient to protect from lethal challenge with DENV-1, DENV-2 or DENV-4. In non-human primates, administration of TDV induces innate immune responses as well as long lasting antibody and cellular immunity. In Phase 1 clinical trials, the safety and immunogenicity of two different formulations were assessed after intradermal or subcutaneous administration to healthy, flavivirus-naïve adults. TDV administration was generally well-tolerated independent of dose and route. The vaccine induced neutralizing antibody responses to all four DENV serotypes: after a single administration of the higher formulation, 24–67%% of the subjects seroconverted to all four DENV and >80% seroconverted to three or more viruses. In addition, TDV induced CD8⁺ T cell responses to the non-structural NS1, NS3 and NS5 proteins of DENV. TDV has been also shown to be generally well tolerated and immunogenic in a Phase 2 clinical trial in dengue endemic countries in adults and children as young as 18 months. Additional clinical studies are ongoing in preparation for a Phase 3 safety and efficacy study.     

Lot-to-lot consistency of a tetravalent dengue vaccine in healthy adults in Australia: A randomised study

BackgroundThe recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) has undergone extensive clinical trials. Here safety and consistency of immunogenicity of phase III manufacturing lots of CYD-TDV were evaluated and compared with a phase II lot and placebo in a dengue-naïve population.MethodsHealthy 18–60 year-olds were randomly assigned in a 3:3:3:3:1 ratio to receive three subcutaneous doses of either CYD-TDV from any one of three phase III lots or a phase II lot, or placebo, respectively in a 0, 6, 12 month dosing schedule. Neutralising antibody geometric mean titres (PRNT₅₀ GMTs) for each of the four dengue serotypes were compared in sera collected 28 days after the third vaccination—equivalence among lots was demonstrated if the lower and upper limits of the two-sided 95% CIs of the GMT ratio were ≥0.5 and ≤2.0, respectively.Results712 participants received vaccine or placebo and 614 (86%) completed the study; 17 (2.4%) participants withdrew after adverse events. Equivalence of phase III lots was demonstrated for 11 of 12 pairwise comparisons. One of three comparisons for serotype 2 was not statistically equivalent. GMTs for serotype 2 in phase III lots were close to each other (65.9, 44.1 and 58.1, respectively).ConclusionsPhase III lots can be produced in a consistent manner with predictable immune response and acceptable safety profile similar to previously characterised phase II lots. The phase III lots may be considered as not clinically different as statistical equivalence was shown for serotypes 1, 3 and 4 across the phase III lots. For serotype 2, although equivalence was not shown between two lots, the GMTs observed in the phase III lots were consistently higher than those for the phase II lot. As such, in our view, biological equivalence for all serotypes was demonstrated.     

Impact of anti-orthopoxvirus neutralizing antibodies induced by a heterologous prime-boost HIV-1 vaccine on insert-specific immune responses

Background

The impact of anti-vector immunity on the elicitation of insert-specific immune responses is important to understand in vaccine development. HVTN 055 was a 150 person phase I randomized, controlled HIV vaccine trial of recombinant modified vaccinia Ankara (rMVA) and fowlpox (rFPV) with matched HIV-1 inserts which demonstrated increased CD8+ T-cell immune responses in the heterologous vaccine group. The controls used in this study were the empty vectors (MVA and FPV).

Methods

Anti-MVA and anti-vaccinia neutralizing antibodies (NAbs) were measured and compared with cellular and humoral HIV-1-specific immune responses.

Results

Elicitation of anti-vector responses increased with increasing dose of MVA and up to 2 administrations. Further inoculations of MVA (up to 5) did not increase the magnitude of the anti-MVA response but did delay the anti-vector NAb titre decay. There was no evidence that the insert impaired the anti-vector response, nor that anti-vector immunity attenuated the insert-specific responses.

Conclusion

Two doses of MVA may be ideal for the elicitation of orthopoxvirus immune responses with further doses maintaining increased titres against the vector. We found no evidence that eliciting HIV insert- or MVA vector-specific immune responses interfered with elicitation of immune responses to the other.     

Evaluating functional antibodies in rhesus monkeys immunized with hepatitis B virus surface antigen vaccine with novel adjuvant formulations

Effective and safe novel adjuvants are of great interest to the vaccine research community. In this study, we describe our evaluation of adjuvant formulations containing a TLR9 agonist adjuvant (ISS1018) or ISCOMATRIX™ adjuvant for a two-dose regimen of hepatitis B virus surface antigen virus-like particle vaccine in mice and rhesus macaques. Our results show a 10-20 fold improvement in Ab binding titers determined in an antigen-sandwich assay for adjuvant formulations with ISCOMATRIX™ adjuvant, in comparison to routine aluminum formulation. Furthermore, we optimized a competition assay to evaluate a functional component of immune sera, using a conformation-dependent and protective mAb, RFHBs1, as the probe. Although good correlation was observed between Ab binding titers from the antigen-sandwich assay and functional titers from the in-solution competition against RFHBs1, the latter assessment provided a much more stringent ranking of adjuvant formulations than the former. These results indicate the importance of evaluating functional Abs when assessing and comparing novel adjuvant formulations, as it provides another angle to investigate the effects of change in adjuvant composition on antigenic integrity of the testing vaccines.     

A multivalent polyomavirus vaccine elicits durable neutralizing antibody responses in macaques

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of $\ge$ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.     

三硝基甲苯对恒河猴的亚慢性毒性研究

用60mg/kg和120mg/kgTNT给恒河猴灌胃三个月。主要引起血液中高铁血红蛋白形成,变性珠蛋白小体出现和网织红细胞升高,继而引起脾脏和骨髓相应改变,肝线粒体脂质过氧化物含量增高和血清胆酸一时性增高,晶状体未见明显改变。说明在本实验条件下,TNT对恒河猴血液系统的毒性明显,对肝脏影响不明显,对晶状体则未见影响。     

Induction of T lymphocyte responses to dengue virus by a candidate tetravalent live attenuated dengue virus vaccine

Development of a safe and immunogenic tetravalent dengue virus (DV) vaccine has been designated as a priority by the World Health Organization. We characterized the T cell response to DV induced by a candidate live attenuated tetravalent DV vaccine as part of a phase I study. Proliferation and cytotoxic T lymphocyte (CTL) responses to multiple DV serotypes were detected in six of six and four of four subjects studied, respectively. Proliferation responses were higher to DV serotypes 1 and 3 than to serotypes 2 and 4. CTL responses were higher to DV serotypes 2 and 3 than to serotype 1, and included serotype cross-reactive responses. Production of interferon-γ, but not IL-4, was observed in response to DV stimulation. This candidate vaccine is immunogenic for both CD4+ and CD8+ T lymphocytes. However, T cell responses to the four DV serotypes were not equivalent, suggesting that the vaccine could be further optimized.