Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

Persistence of immunity and impact of third dose of inactivated COVID-19 vaccine against emerging variants.

This is a comprehensive report on immunogenicity of COVAXIN® booster dose against ancestral and Variants of Concern (VOCs) up to 12 months. It is well known that neutralizing antibodies induced by COVID-19 vaccines wane within 6 months of vaccination leading to questions on the effectiveness of two-dose vaccination against breakthrough infections. Therefore, we assessed the persistence of immunogenicity up to 6 months after a two or three-dose with BBV152 and the safety of a booster dose in an ongoing phase 2, double-blind, randomized controlled trial (ClinicalTrials.gov: NCT04471519). We report persistence of humoral and cell mediated immunity up to 12 months of vaccination, despite decline in the magnitude of antibody titers. Administration of a third dose of BBV152 increased neutralization titers against both homologous (D614G) and heterologous strains (Alpha, Beta, Delta, Delta Plus and Omicron) with a slight increase in B cell memory responses. Thus, seronversion rate remain high in boosted recipients compared to non-booster, even after 6 months, post third dose against variants. No serious adverse events observed, except pain at the injection site, itching and redness. Hence, these results indicate that a booster dose of BBV152 is safe and necessary to ensure persistent immunity to minimize breakthrough infections of COVID-19, due to newly emerging variants.Trial registration: Registered with the Clinical Trials Registry (India) No. CTRI/2021/04/032942, dated 19/04/2021 and on Clinicaltrials.gov: NCT04471519.

Immunogenicity and reactogenicity of an inactivated SARS-CoV-2 vaccine (BBV152) in children aged 2-18 years: interim data from an open-label, non-randomised, age de-escalation phase 2/3 study.

BACKGROUND: Despite having milder symptoms than adults, children are still susceptible to and can transmit SARS-CoV-2. Vaccination across all age groups is therefore necessary to curtail the pandemic. Among the available COVID-19 vaccine platforms, an inactivated vaccine platform has the advantage of excellent safety profile across all age groups; hence, we conducted an age de-escalation study to assess the safety, reactogenicity, and immunogenicity of an inactivated COVID-19 vaccine, BBV152 (COVAXIN; Bharat Biotech International, Hyderabad, India), in children aged 2-18 years. METHODS: In this phase 2/3 open-label, non-randomised, multicentre study done in six hospitals in India, healthy children (male or female) aged 2-18 years were eligible for inclusion into the study. Children who had positive SARS-CoV-2 nucleic acid and serology tests at baseline, or any history of previous SARS-CoV-2 infection, or with known immunosuppressive condition were excluded. Children were sequentially enrolled into one of three groups (>12 to ≤18 years [group 1], >6 to 12 years [group 2], or ≥2 to 6 years [group 3]) and administered with adult formulation of BBV152 as two 0·5 mL intramuscular doses on days 0 and 28. Co-primary endpoints were solicited adverse events for 7 days post-vaccination and neutralising antibody titres on day 56, 28 days after the second dose. Immunogenicity endpoints were compared with Biodefense and Emerging Infections, Research Resources Repository (BEI) reference serum samples and from adults who received two doses of BBV152 in the same schedule in a previously reported phase 2 study. The trial is registered with the Clinical Trials Registry, India (CTRI/2021/05/033752) and ClinicalTrials.gov (NCT04918797). FINDINGS: From May 27, 2021, to July 10, 2021, we enrolled 526 children sequentially into groups 1 (n=176), 2 (n=175), and 3 (n=175). Vaccination was well tolerated, with no differences in reactogenicity between the three age groups, and no serious adverse events, deaths, or withdrawals due to an adverse event. Local reactions mainly consisted of mild injection site pain in 46 (26%) of 176 participants in group 1, 61 (35%) of 175 in group 2, and 39 (22%) of 175 in group 3 after dose 1; and 39 (22%) of 176 in group 1, 43 of 175 (25%) in group 2, and 14 of 175 (8%) in group 3 after dose 2; there were no cases of severe pain and few reports of other local reactions. After dose 1, the most frequent solicited systemic adverse event was mild-to-moderate fever, reported in eight (5%) of 176 participants in group 1, 17 (10%) of 175 in group 2, and 22 (13%) of 175 in group 3. No case of severe fever was reported, and rates of all fever were all 4% or less after dose 2. Geometric mean titres (GMTs) of microneutralisation antibodies at day 56 in groups 1 (138·8 [95% CI 111·0-173·6]), 2 (137·4 [99·1-167·5]), and 3 (197·6 [176·4-221·4]) were similar to titres in vaccinated adults (160·1 [135·8-188·8]) and with BEI reference serum samples (103·3 [50·3-202·1]). Similar results were obtained using the plaque reduction neutralisation test (PRNT), in which 166 (95%) of 175 participants in group 1, 165 (98%) of 168 in group 2, and 169 (98%) of 172 in group 3 seroconverted at day 56. The GMT ratio of PRNT titres in children and adults was 1·76 (95% CI 1·32-2·33), indicating a superior response in children compared with adults. INTERPRETATION: BBV152 was well tolerated in children aged 2-18 years, and induced higher neutralising antibody responses than those observed in adults, in whom the efficacy (ie, the prevention or decrease in the severity of COVID-19 infection) has been demonstrated. FUNDING: Bharat Biotech International.

Efficacy, safety, and lot-to-lot immunogenicity of an inactivated SARS-CoV-2 vaccine (BBV152): interim results of a randomised, double-blind, controlled, phase 3 trial.

BACKGROUND: We report the clinical efficacy against COVID-19 infection of BBV152, a whole virion inactivated SARS-CoV-2 vaccine formulated with a toll-like receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG) in Indian adults. METHODS: We did a randomised, double-blind, placebo-controlled, multicentre, phase 3 clinical trial in 25 Indian hospitals or medical clinics to evaluate the efficacy, safety, and immunological lot consistency of BBV152. Adults (age ≥18 years) who were healthy or had stable chronic medical conditions (not an immunocompromising condition or requiring treatment with immunosuppressive therapy) were randomised 1:1 with a computer-generated randomisation scheme (stratified for the presence or absence of chronic conditions) to receive two intramuscular doses of vaccine or placebo administered 4 weeks apart. Participants, investigators, study coordinators, study-related personnel, the sponsor, and nurses who administered the vaccines were masked to treatment group allocation; an unmasked contract research organisation and a masked expert adjudication panel assessed outcomes. The primary outcome was the efficacy of the BBV152 vaccine in preventing a first occurrence of laboratory-confirmed (RT-PCR-positive) symptomatic COVID-19 (any severity), occurring at least 14 days after the second dose in the per-protocol population. We also assessed safety and reactogenicity throughout the duration of the study in all participants who had received at least one dose of vaccine or placebo. This report contains interim results (data cutoff May 17, 2021) regarding immunogenicity and safety outcomes (captured on days 0 to 56) and efficacy results with a median of 99 days for the study population. The trial was registered on the Indian Clinical Trials Registry India, CTRI/2020/11/028976, and ClinicalTrials.gov, NCT04641481 (active, not recruiting). FINDINGS: Between Nov 16, 2020, and Jan 7, 2021, we recruited 25 798 participants who were randomly assigned to receive BBV152 or placebo; 24 419 received two doses of BBV152 (n=12 221) or placebo (n=12 198). Efficacy analysis was dependent on having 130 cases of symptomatic COVID-19, which occurred when 16 973 initially seronegative participants had at least 14 days follow-up after the second dose. 24 (0·3%) cases occurred among 8471 vaccine recipients and 106 (1·2%) among 8502 placebo recipients, giving an overall estimated vaccine efficacy of 77·8% (95% CI 65·2-86·4). In the safety population (n=25 753), 5959 adverse events occurred in 3194 participants. BBV152 was well tolerated; the same proportion of participants reported adverse events in the vaccine group (1597 [12·4%] of 12 879) and placebo group (1597 [12·4%] of 12 874), with no clinically significant differences in the distributions of solicited, unsolicited, or serious adverse events between the groups, and no cases of anaphylaxis or vaccine-related deaths. INTERPRETATION: BBV152 was highly efficacious against laboratory-confirmed symptomatic COVID-19 disease in adults. Vaccination was well tolerated with no safety concerns raised in this interim analysis. FUNDING: Bharat Biotech International and Indian Council of Medical Research.

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV152: interim results from a double-blind, randomised, multicentre, phase 2 trial, and 3-month follow-up of a double-blind, randomised phase 1 trial.

BACKGROUND: BBV152 is a whole-virion inactivated SARS-CoV-2 vaccine (3 µg or 6 µg) formulated with a toll-like receptor 7/8 agonist molecule (IMDG) adsorbed to alum (Algel). We previously reported findings from a double-blind, multicentre, randomised, controlled phase 1 trial on the safety and immunogenicity of three different formulations of BBV152 (3 µg with Algel-IMDG, 6 µg with Algel-IMDG, or 6 µg with Algel) and one Algel-only control (no antigen), with the first dose administered on day 0 and the second dose on day 14. The 3 µg and 6 µg with Algel-IMDG formulations were selected for this phase 2 study. Herein, we report interim findings of the phase 2 trial on the immunogenicity and safety of BBV152, with the first dose administered on day 0 and the second dose on day 28. METHODS: We did a double-blind, randomised, multicentre, phase 2 clinical trial to evaluate the immunogenicity and safety of BBV152 in healthy adults and adolescents (aged 12-65 years) at nine hospitals in India. Participants with positive SARS-CoV-2 nucleic acid and serology tests were excluded. Participants were randomly assigned (1:1) to receive either 3 µg with Algel-IMDG or 6 µg with Algel-IMDG. Block randomisation was done by use of an interactive web response system. Participants, investigators, study coordinators, study-related personnel, and the sponsor were masked to treatment group allocation. Two intramuscular doses of vaccine were administered on day 0 and day 28. The primary outcome was SARS-CoV-2 wild-type neutralising antibody titres and seroconversion rates (defined as a post-vaccination titre that was at least four-fold higher than the baseline titre) at 4 weeks after the second dose (day 56), measured by use of the plaque-reduction neutralisation test (PRNT50) and the microneutralisation test (MNT50). The primary outcome was assessed in all participants who had received both doses of the vaccine. Cell-mediated responses were a secondary outcome and were assessed by T-helper-1 (Th1)/Th2 profiling at 2 weeks after the second dose (day 42). Safety was assessed in all participants who received at least one dose of the vaccine. In addition, we report immunogenicity results from a follow-up blood draw collected from phase 1 trial participants at 3 months after they received the second dose (day 104). This trial is registered at ClinicalTrials.gov, NCT04471519. FINDINGS: Between Sept 5 and 12, 2020, 921 participants were screened, of whom 380 were enrolled and randomly assigned to the 3 µg with Algel-IMDG group (n=190) or 6 µg with Algel-IMDG group (n=190). Geometric mean titres (GMTs; PRNT50) at day 56 were significantly higher in the 6 µg with Algel-IMDG group (197·0 [95% CI 155·6-249·4]) than the 3 µg with Algel-IMDG group (100·9 [74·1-137·4]; p=0·0041). Seroconversion based on PRNT50 at day 56 was reported in 171 (92·9% [95% CI 88·2-96·2] of 184 participants in the 3 µg with Algel-IMDG group and 174 (98·3% [95·1-99·6]) of 177 participants in the 6 µg with Algel-IMDG group. GMTs (MNT50) at day 56 were 92·5 (95% CI 77·7-110·2) in the 3 µg with Algel-IMDG group and 160·1 (135·8-188·8) in the 6 µg with Algel-IMDG group. Seroconversion based on MNT50 at day 56 was reported in 162 (88·0% [95% CI 82·4-92·3]) of 184 participants in the 3 µg with Algel-IMDG group and 171 (96·6% [92·8-98·8]) of 177 participants in the 6 µg with Algel-IMDG group. The 3 µg with Algel-IMDG and 6 µg with Algel-IMDG formulations elicited T-cell responses that were biased to a Th1 phenotype at day 42. No significant difference in the proportion of participants who had a solicited local or systemic adverse reaction in the 3 µg with Algel-IMDG group (38 [20·0%; 95% CI 14·7-26·5] of 190) and the 6 µg with Algel-IMDG group (40 [21·1%; 15·5-27·5] of 190) was observed on days 0-7 and days 28-35; no serious adverse events were reported in the study. From the phase 1 trial, 3-month post-second-dose GMTs (MNT50) were 39·9 (95% CI 32·0-49·9) in the 3µg with Algel-IMDG group, 69·5 (53·7-89·9) in the 6 µg with Algel-IMDG group, 53·3 (40·1-71·0) in the 6 µg with Algel group, and 20·7 (14·5-29·5) in the Algel alone group. INTERPRETATION: In the phase 1 trial, BBV152 induced high neutralising antibody responses that remained elevated in all participants at 3 months after the second vaccination. In the phase 2 trial, BBV152 showed better reactogenicity and safety outcomes, and enhanced humoral and cell-mediated immune responses compared with the phase 1 trial. The 6 µg with Algel-IMDG formulation has been selected for the phase 3 efficacy trial. FUNDING: Bharat Biotech International. TRANSLATION: For the Hindi translation of the abstract see Supplementary Materials section.

Th1 skewed immune response of whole virion inactivated SARS CoV 2 vaccine and its safety evaluation.

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated (WVI) SARS-CoV-2 vaccine (BBV152), adjuvanted with aluminum hydroxide gel (Algel), or TLR7/8 agonist chemisorbed Algel. We used a well-characterized SARS-CoV-2 strain and an established Vero cell platform to produce large-scale GMP-grade highly purified inactivated antigen. Product development and manufacturing process were carried out in a BSL-3 facility. Immunogenicity and safety were determined at two antigen concentrations (3µg and 6µg), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers (NAb), at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation contains TLR7/8 agonist adjuvant-induced Th1-biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2-specific IFN-γ+ CD4+ T lymphocyte response. Our results support further development for phase I/II clinical trials in humans.

Immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidate, BBV152 in rhesus macaques.

The COVID-19 pandemic is a global health crisis that poses a great challenge to the public health system of affected countries. Safe and effective vaccines are needed to overcome this crisis. Here, we develop and assess the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine in rhesus macaques. Twenty macaques were divided into four groups of five animals each. One group was administered a placebo, while three groups were immunized with three different vaccine candidates of BBV152 at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which exhibited interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. This vaccine candidate BBV152 has completed Phase I/II (NCT04471519) clinical trials in India and is presently in phase III, data of this study substantiates the immunogenicity and protective efficacy of the vaccine candidates.

Immunogenicity and protective efficacy of BBV152, whole virion inactivated SARS- CoV-2 vaccine candidates in the Syrian hamster model.

The availability of a safe and effective vaccine would be the eventual measure to deal with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) threat. Here, we have assessed the immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidates BBV152A, BBV152B, and BBV152C in Syrian hamsters. Three dose vaccination regimes with vaccine candidates induced significant titers of SARS-CoV-2-specific IgG and neutralizing antibodies. BBV152A and BBV152B vaccine candidates remarkably generated a quick and robust immune response. Post-SARS-CoV-2 infection, vaccinated hamsters did not show any histopathological changes in the lungs. The protection of the hamster was evident by the rapid clearance of the virus from lower respiratory tract, reduced virus load in upper respiratory tract, absence of lung pathology, and robust humoral immune response. These findings confirm the immunogenic potential of the vaccine candidates and further protection of hamsters challenged with SARS-CoV-2. Of the three candidates, BBV152A showed the better response.

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine, BBV152: a double-blind, randomised, phase 1 trial.

BACKGROUND: To mitigate the effects of COVID-19, a vaccine is urgently needed. BBV152 is a whole-virion inactivated SARS-CoV-2 vaccine formulated with a toll-like receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG) or alum (Algel). METHODS: We did a double-blind, multicentre, randomised, controlled phase 1 trial to assess the safety and immunogenicity of BBV152 at 11 hospitals across India. Healthy adults aged 18-55 years who were deemed healthy by the investigator were eligible. Individuals with positive SARS-CoV-2 nucleic acid and/or serology tests were excluded. Participants were randomly assigned to receive either one of three vaccine formulations (3 µg with Algel-IMDG, 6 µg with Algel-IMDG, or 6 µg with Algel) or an Algel only control vaccine group. Block randomisation was done with a web response platform. Participants and investigators were masked to treatment group allocation. Two intramuscular doses of vaccines were administered on day 0 (the day of randomisation) and day 14. Primary outcomes were solicited local and systemic reactogenicity events at 2 h and 7 days after vaccination and throughout the full study duration, including serious adverse events. Secondary outcome was seroconversion (at least four-fold increase from baseline) based on wild-type virus neutralisation. Cell-mediated responses were evaluated by intracellular staining and ELISpot. The trial is registered at ClinicalTrials.gov (NCT04471519). FINDINGS: Between July 13 and 30, 2020, 827 participants were screened, of whom 375 were enrolled. Among the enrolled participants, 100 each were randomly assigned to the three vaccine groups, and 75 were randomly assigned to the control group (Algel only). After both doses, solicited local and systemic adverse reactions were reported by 17 (17%; 95% CI 10·5-26·1) participants in the 3 µg with Algel-IMDG group, 21 (21%; 13·8-30·5) in the 6 µg with Algel-IMDG group, 14 (14%; 8·1-22·7) in the 6 µg with Algel group, and ten (10%; 6·9-23·6) in the Algel-only group. The most common solicited adverse events were injection site pain (17 [5%] of 375 participants), headache (13 [3%]), fatigue (11 [3%]), fever (nine [2%]), and nausea or vomiting (seven [2%]). All solicited adverse events were mild (43 [69%] of 62) or moderate (19 [31%]) and were more frequent after the first dose. One serious adverse event of viral pneumonitis was reported in the 6 µg with Algel group, unrelated to the vaccine. Seroconversion rates (%) were 87·9, 91·9, and 82·8 in the 3 µg with Algel-IMDG, 6 µg with Algel-IMDG, and 6 µg with Algel groups, respectively. CD4+ and CD8+ T-cell responses were detected in a subset of 16 participants from both Algel-IMDG groups. INTERPRETATION: BBV152 led to tolerable safety outcomes and enhanced immune responses. Both Algel-IMDG formulations were selected for phase 2 immunogenicity trials. Further efficacy trials are warranted. FUNDING: Bharat Biotech International.

Coronavirus Vaccine: Light at the End of the Tunnel.

The world is currently facing an unprecedented global pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Predicting the next source of the pandemic can be very challenging. As vaccination is the best way to prevent an infectious disease, the development of an effective vaccine against SARS-CoV-2 can not only reduce the morbidity and mortality associated with it, but can also lessen the economic impact. As the traditional method of vaccine development takes many years for a vaccine to be available to the society, the vaccine development for SARS-CoV-2 should be speeded up using a pandemic approach with fast-track approvals from the regulatory authorities. Various challenges associated with developing a vaccine during the pandemic such as technological hurdles, clinical development pathways, regulatory issues, and support from global funding agencies are expressed here.

Immunogenicity of a Candidate Ebola Hemorrhagic Fever Vaccine in Mice Based on Controlled In Vitro Expression of Ebolavirus Glycoprotein.

Ebolavirus (EBOV) is the etiology of Ebola hemorrhagic fever (EHF). A major EHF outbreak in 2014-2015 in West Africa claimed >11,000 lives. A licensed vaccine is not available for EHF, although several vaccines have undergone clinical trials. We developed a human adenovirus (Ad) serotype 5-based candidate EHF vaccine based on controlled expression of the EBOV (Makona strain) glycoprotein (GP) as the immunogen. Two clones, AdGP72 and AdGP75, and a control Ad515 vector, were generated and tested for protein expression in vitro and immunogenicity in mice. Eight groups of mice were immunized with three doses of buffer, Ad515, AdGP72, and AdGP75, by two different dose regimens. Three different antigens (AdGP75-infected Vero E6 cell extract and two baculovirus expressed EBOV GP antigens, namely, GP alone or GP with EBOV VP40) were used to evaluate the immune response. Expression studies indicated that full-length GP was cleaved into its component subunits when expressed in mammalian cells through the Ad vectors. Moreover, in coimmunoprecipitation studies, EBOV GP was found to be associated with VP40 when expressed in baculoviruses. The candidate vaccines were immunogenic in mice, as evaluated by enzyme-linked immunosorbent assay using mammalian- or baculovirus-derived antigens. Further characterization and development of the candidate vaccines are warranted.

Immunogenicity and Induction of Functional Antibodies in Rabbits Immunized with a Trivalent Typhoid-Invasive Nontyphoidal Salmonella Glycoconjugate Formulation.

Typhoid fever due to Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) infections caused by serovars Enteritidis (SE) and Typhimurium (STm) are major pediatric health problems in sub-Saharan Africa. Typhoid has high complication rates, and iNTS infections have high case fatality rates; moreover, emerging antimicrobial resistance is diminishing treatment options. Vi capsule-based typhoid conjugate vaccine (Typbar-TCV™), licensed in India and pre-qualified by the World Health Organization, elicits durable immunity when administered to infants, but no iNTS vaccines are licensed or imminent. We have developed monovalent SE and STm glycoconjugate vaccines based on coupling lipopolysaccharide-derived core-O polysaccharide (COPS) to phase 1 flagellin protein (FliC) from the homologous serovar. Herein, we report the immunogenicity of multivalent formulations of iNTS COPS:FliC conjugates with Typbar-TCV™. Rabbits immunized with the trivalent typhoid-iNTS glycoconjugate vaccine generated high titers of serum IgG antibody to all three polysaccharide antigens for which anti-COPS IgG antibodies were directed primarily against serogroup-specific OPS epitopes. Responses to SE and STm FliC were lower relative to anti-COPS titers. Post-vaccination rabbit sera mediated bactericidal activity in-vitro, and protected mice after passive transfer against challenge with virulent SE or STm Malian blood isolates. These results support accelerated progression to clinical trials.

Protective efficacy of Zika vaccine in AG129 mouse model.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes asymptomatic infection or presents only mild symptoms in majority of those infected. However, vaccination for ZIKV is a public health priority due to serious congenital and neuropathological abnormalities observed as a sequelae of the virus infection in the recent epidemics. We have developed an inactivated virus vaccine with the African MR 766 strain. Here we show that two doses of the vaccine provided 100% efficacy against mortality and disease following challenge with homotypic MR 766 and the heterotypic FSS 13025 ZIKV strains in the Type I and Type II interferon deficient AG129 mice. Two doses of the vaccine elicited high titer of neutralizing antibodies in Balb/c mice, and the vaccine antisera conferred protection against virus challenge in passively immunized mice. The studies were useful to rationalize vaccine doses for protective efficacy. Furthermore, the vaccine antisera neutralized the homotypic and heterotypic ZIKV strains in vitro with equivalent efficiency. Our study suggests a single ZIKV serotype, and that the development of an effective vaccine may not be limited by the choice of virus strain.

Safety and immunogenicity of a Vi polysaccharide-tetanus toxoid conjugate vaccine (Typbar-TCV) in healthy infants, children, and adults in typhoid endemic areas: a multicenter, 2-cohort, open-label, double-blind, randomized controlled phase 3 study.

BACKGROUND: Enteric fever caused by Salmonella Typhi remains a major public health problem in developing countries. Typbar-TCV is a single-dose typhoid Vi polysaccharide-tetanus toxoid conjugate vaccine for persons ≥6 months of age. METHODS: Six hundred fifty-four healthy subjects aged 2-45 years enrolled in a double-blind, randomized controlled trial (RCT) received a single dose of Typbar-TCV or comparator "Vi polysaccharide" (Typbar), and 327 healthy subjects aged 6-23 months received a single dose of Typbar-TCV in an open-label trial (OLT); both received single- or multidose presentations from different lots. After 2 years, subsets in each group received a booster dose. The primary objective included analysis of geometric mean titer (GMTs) and 4-fold rise of anti-Vi serum immunoglobulin G (IgG) enzyme-linked immunosorbent assay titers over baseline (seroconversion [SCN]) 42 days after immunization. RESULTS: Typbar-TCV recipients in the RCT attained higher anti-Vi IgG GMTs 42 days after immunization (SCN, 97%; GMT, 1293 [95% confidence interval {CI}, 1153-1449]) than recipients of Typbar (SCN, 93%; GMT, 411 [95% CI, 359-471]) (P < .001). Typbar-TCV was highly immunogenic in the OLT (SCN, 98%; GMT, 1937 [95% CI, 1785-2103]). Two years after vaccination, anti-Vi titers remained higher in Typbar-TCV subjects (GMT, 82 [95% CI, 73-92]); and exhibited higher avidity (geometric mean avidity index [GMAI], 60%) than in Typbar recipients (GMT, 46 [95% CI, 40-53]; GMAI 46%) in the RCT (P < .001). OLT Typbar-TCV recipients achieved GMT of 48 (95% CI, 42-55) and GMAI of 57%. Typbar-TCV induced multiple IgG subclasses and strong booster responses in all ages. No serious vaccine-attributable adverse events were observed. CONCLUSIONS: Single-dose Typbar-TCV is well tolerated and induces robust and long-lasting serum anti-Vi IgG across age groups. CLINICAL TRIALS REGISTRATION: CTRI/2011/08/001957, CTRI/2014/01/004341.

A Japanese Encephalitis Vaccine From India Induces Durable and Cross-protective Immunity Against Temporally and Spatially Wide-ranging Global Field Strains.

BACKGROUND: Japanese encephalitis (JE) is a vaccine-preventable acute disease. We report the results of a phase 2/3 trial of JENVAC, a Vero cell-derived vaccine developed using an Indian strain of JE virus (JEV). METHODS: JENVAC was administered in 2 doses 28 days apart, and immunogenicity was compared to that from a single dose of SA-14-14-2, the only approved JE vaccine and regimen at the time in India. RESULTS: After both the doses, seroconversion and seroprotection were >90% for JENVAC. For SA-14-14-2, seroconversion and seroprotection were 57.69% and 77.56%, respectively, on day 28 and 39.74% and 60.26%, respectively, on day 56. The geometric mean titers at day 28 and day 56 were 145.04 and 460.53, respectively, for JENVAC and 38.56 and 25.29, respectively, for SA-14-14-2. With a single dose of JENVAC, seroprotection titers lasted at least 12 months in >80% of the subjects. Following receipt of 2 doses, 61.17% of subjects retained seroprotection titers at 24 months, and immunogenicity criteria were higher than that for SA-14-14-2 at 12, 18, and 24 months each. Sera from JENVAC subjects neutralized JEV genotypes I, II, III, and IV equally well. Adverse events were not significantly different between the 2 vaccines. CONCLUSIONS: JENVAC elicits long-lasting, broadly protective immunity. CLINICAL TRIALS REGISTRATION: CTRI/2011/07/001855.

Evaluation of safety and immunogenicity of HNVAC, an MDCK-based H1N1 pandemic influenza vaccine, in Phase I single centre and Phase II/III multi-centre, double-blind, randomized, placebo-controlled, parallel assignment studies.

The clinical evaluation of the MDCK-based H1N1 pandemic influenza vaccine HNVAC in adults aged 18-65 years is reported. In the Phase I randomized, double-blind, placebo-controlled, single-centre study, 160 subjects were parallelly assigned 3:1 to vaccine:placebo groups (n=60:20) with both the aluminium hydroxide adjuvanted and non-adjuvanted vaccine formulations. A single dose of both the formulations containing 15 µg of haemagglutinin protein showed minimal adverse reactions, the most common of which were pain at injection site (11.67%) and fever (10.00%). Both formulations produced 74-81% seroprotection (SRP: titre of ≥40), 67-70% seroconversion (SRC: four-fold increase in titres between days 0 and 21), and a four-fold increase in geometric mean titres (GMT). Aluminium hydroxide did not have a significant effect either on immunogenicity or on reactogenicity. Nevertheless, based on its recognized positive effects on the stability and immunogenicity of many vaccines, and its marginal benefit in both pre-clinical and Phase I studies of HNVAC, alum adjuvanted HNVAC was further tested in a staggered Phase II/III randomized, double-blind, placebo-controlled, multi-centre study of 200 and 195 subjects, respectively, parallelly assigned 4:1 to adjuvanted vaccine and placebo groups. In these studies, the most common adverse reactions were pain at injection site (6.88% and 5.77% in Stage 1 and Stage 2, respectively) and fever (7.50% and 7.05%, respectively), and a single dose resulted in 87-90% SRP, 85-86% SRC, and a nearly six-fold increase in GMT, meeting or exceeding licensing criteria. It is concluded that HNVAC is safe and immunogenic to adults of 18-65 years.

Development and preclinical testing of HNVAC, a cell culture-based H1N1 pandemic influenza vaccine from India.

Several limitations of the use of embryonated eggs and the threat of pandemics have highlighted the need for other platforms for the production of influenza vaccines. We report the indigenous development and pre-clinical testing of an MDCK-based H1N1 pandemic influenza vaccine HNVAC from India. The cell bank and virus seed were characterized extensively. The cells were characterized by PCR, electron microscopy, and karyotyping, and found to be of female canine epithelial origin. The virus was confirmed by neutralization, haemagglutination inhibition, neuraminidase inhibition, and PCR and nucleotide sequencing. Adventitious agent testing was performed by both in vitro and in vivo studies. The in vitro studies included culturing, haemadsorption, haemagglutination, PCR and RT-PCR, whereas in vivo studies included passage in embryonated eggs and in laboratory animals. Both cell bank and virus seed were free of adventitious agents. MDCK cell lysates as well as cellular DNA did not produce tumours in newborn or adult laboratory animals. The bioprocess parameters were standardized to recover antigen with minimal levels of process-related impurities. The vaccine bulk was tested for the presence of specific antigen, and quantified by single radial immunodiffusion. Finally, non-adjuvanted and aluminium hydroxide adjuvanted vaccine formulations were found to be safe in preclinical toxicity studies in mice, rats, guinea pigs and rabbits, and immunogenic in mice and rabbits. This is the first and only cell culture-based influenza vaccine platform developed in any developing country.

Genetic diversity of Chikungunya virus, India 2006-2010: evolutionary dynamics and serotype analyses.

The genetic diversity of Chikungunya virus (CHIKV) causing recurring outbreaks in India since 2006 was studied. The 2006 epidemic was caused by a virus strain of the East, Central and South African (ECSA) genotype with 226A in the E1 glycoprotein. The variant strain with E1-A226V mutation caused outbreaks since 2007 in the state of Kerala where Aedes albopictus is the abundant mosquito vector. Molecular epidemiology data since 2007 is scarce from other regions of the country. RT-PCR, sequencing and phylogenetic analyses of CHIKV isolates from the 2009 to 2010 epidemics in the States of Tamil Nadu and Andhra Pradesh placed them in a separate clade within the ECSA lineage. The isolates of the study had 226A in the E1 glycoprotein. The isolates had a novel E1-K211E mutation that was under significant positive selection. E1-211E is highly conserved in the Asian genotype of the virus circulated by Aedes aegypti. Unique mutations in E2 glycoprotein were identified. The two sub-lineages of ECSA genotype circulating in India parallel the abundance of Ae. albopictus and Ae. aegypti. Novel mutations in the envelope glycoproteins suggest adaptive evolution of the virus to local vector abundance. Cross neutralization of the virus isolates from recurring Indian epidemics indicated that no distinct serotypes had evolved. The study has provided insights into the origin, distribution and evolutionary adaptation of the virus to local vector abundance in the region that has reportedly, the highest incidence of CHIKV infection in the world.