Puneet Kumar Gupta

The Requirement for Potent Adjuvants To Enhance the Immunogenicity and Protective Efficacy of Protein Vaccines Can Be Overcome by Prior Immunization with a Recombinant Adenovirus

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5–protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Parity and placental infection affect antibody responses against Plasmodium falciparum during pregnancy.

ABSTRACT Women are at higher risk of Plasmodium falciparum infection when pregnant. The decreasing risk of malaria with subsequent pregnancies is attributed to parity-dependent acquisition of antibodies against placental parasites expressing variant surface antigens, VAR2CSA, that mediate placental sequestration through adhesion to chondroitin sulfate A (CSA). However, modulation of immunity during pregnancy may also contribute to increase the risk of malaria. We compared antibody responses among 30 Mozambican primigravidae and 60 multigravidae at delivery, 40 men, and 40 children. IgG levels were measured against the surface antigens of erythrocytes infected with P. falciparum isolated from 12 pregnant women (4 placental and 8 peripheral blood isolates) and 26 nonpregnant hosts. We also measured IgG levels against merozoite recombinant antigens and total IgG. Placental P. falciparum infection was associated with increased levels of total IgG as well as IgG levels against merozoite antigens and parasite isolates from pregnant and nonpregnant hosts. We therefore stratified comparisons of antibody levels by placental infection. Compared to multigravidae, uninfected primigravidae had lower total IgG as well as lower levels of IgGs against peripheral blood isolates from both pregnant and nonpregnant hosts. These differences were not explained by use of bed nets, season at delivery, neighborhood of residence, or age. Compared to men, infected primigravidae had higher levels of IgGs against isolates from pregnant women and CSA-binding lines but not against other isolates, supporting the concept of a pregnancy-specific development of immunity to these parasite variants. Results of this study show that parity and placental infection can modulate immune responses during pregnancy against malaria parasites.

Induction of humoral immune response against PfMSP-119 and PvMSP-119 using gold nanoparticles along with alum

The C-terminal 19 kDa fragment of merozoite surface protein 1(MSP-1(19)) is an important vaccine candidate but a poor immunogen with available human compatible adjuvants like alum. We have investigated if Gold Nanoparticles (GNPs) based delivery system would enhance immune response to recombinant PfMSP-1(19) and PvMSP-1(19). We found that PfMSP-1(19)/PvMSP-1(19) coated on GNPs or in alum was poorly immunogenic in mice, a robust antibody response was observed when PfMSP-1(19)/PvMSP-1(19) coated GNPs were formulated with alum. PfMSP-1(19)/PvMSP-1(19) coated GNPs strongly recognized conformation specific monoclonal antibodies showing that capping on to GNPs did not compromise with conformational integrity of these antigens, which is critical for the generation of functional antibody response. Anti-PfMSP-1(19) antibodies raised with GNP-alum formulation recognized the native protein on parasite surface and showed invasion inhibition of Plasmodium falciparum parasite in an in vitro assay. Antigen coated GNPs with alum may be useful in the development of an alternate adjuvant formulation.

Improved Pregnancy Outcomes in Women Exposed to Malaria With High Antibody Levels Against Plasmodium falciparum

BACKGROUND Antibodies against VAR2CSA, the Plasmodium falciparum variant surface antigen that binds placental chondroitin sulfate A, have been suggested to mediate protection against malaria in pregnancy but also to be markers of infection. Here, we aimed to identify clinically relevant antibody responses, taking into consideration variations in parasite exposure and human immunodeficiency virus type 1 (HIV) infection status. METHODS Levels of immunoglobulin G (IgG) against placental and pediatric isolates, VAR2CSA (DBL2X, DBL3X, DBL5ε, and DBL6ε domains), and other blood-stage antigens (DBLγ, DBLα, MSP119, AMA1, and EBA175) were measured in plasma specimens from 293 pregnant Mozambican women at delivery. Associations between antibody responses, factors influencing malaria exposure, HIV infection status, and pregnancy outcomes were assessed. RESULTS Maternal antibodies were affected by placental infection, parity, season, and neighborhood of residence. HIV infection modified these associations and attenuated the parity-dependent increase in IgG level. High levels of antibody against AMA1, DBL3X, DBL6ε, placental isolates, and pediatric isolates were associated with increased weight and gestational age of newborns (P ≤ .036) among women with malaria episodes during pregnancy. CONCLUSIONS Antiparasite IgGs in women at delivery are affected by HIV infection, as well as by variations in the exposure to P. falciparum. Heterogeneity of malaria transmission needs to be considered to identify IgGs against VAR2CSA and other parasite antigens associated with improved pregnancy outcomes.

Age-Dependent IgG Subclass Responses to Plasmodium falciparum EBA-175 Are Differentially Associated with Incidence of Malaria in Mozambican Children

ABSTRACT Plasmodium falciparum blood-stage antigens such as merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and the 175-kDa erythrocyte binding antigen (EBA-175) are considered important targets of naturally acquired immunity to malaria. However, it is not clear whether antibodies to these antigens are effectors in protection against clinical disease or mere markers of exposure. In the context of a randomized, placebo-controlled trial of intermittent preventive treatment in infants conducted between 2002 and 2004, antibody responses to Plasmodium falciparum blood-stage antigens in a cohort of 302 Mozambican children were evaluated by immunofluorescence antibody test and enzyme-linked immunosorbent assay at 5, 9, 12, and 24 months of age. We found that IgG subclass responses to EBA-175 were differentially associated with the incidence of malaria in the follow-up period. A double amount of cytophilic IgG1 or IgG3 was associated with a significant decrease in the incidence of malaria (incidence rate ratio [IRR] = 0.49, 95% confidence interval [CI] = 0.25 to 0.97, and P = 0.026 and IRR = 0.44, CI = 0.19 to 0.98, and P = 0.037, respectively), while a double amount of noncytophilic IgG4 was significantly correlated with an increased incidence of malaria (IRR = 3.07, CI = 1.08 to 8.78, P = 0.020). No significant associations between antibodies to the 19-kDa fragment of MSP-1 (MSP-119) or AMA-1 and incidence of malaria were found. Age, previous episodes of malaria, present infection, and neighborhood of residence were the main factors influencing levels of antibodies to all merozoite antigens. Deeper understanding of the acquisition of antibodies against vaccine target antigens in early infancy is crucial for the rational development and deployment of malaria control tools in this vulnerable population.

Phase I Clinical Trial of a Recombinant Blood Stage Vaccine Candidate for Plasmodium falciparum Malaria Based on MSP1 and EBA175

Background A phase I randomised, controlled, single blind, dose escalation trial was conducted to evaluate safety and immunogenicity of JAIVAC-1, a recombinant blood stage vaccine candidate against Plasmodium falciparum malaria, composed of a physical mixture of two recombinant proteins, PfMSP-119, the 19 kD conserved, C-terminal region of PfMSP-1 and PfF2 the receptor-binding F2 domain of EBA175. Method Healthy malaria naïve Indian male subjects aged 18–45 years were recruited from the volunteer database of study site. Fifteen subjects in each cohort, randomised in a ratio of 2:1 and meeting the protocol specific eligibility criteria, were vaccinated either with three doses (10μg, 25μg and 50μg of each antigen) of JAIVAC-1 formulated with adjuvant Montanide ISA 720 or with standard dosage of Hepatitis B vaccine. Each subject received the assigned vaccine in the deltoid muscle of the upper arms on Day 0, Day 28 and Day 180. Results JAIVAC-1 was well tolerated and no serious adverse event was observed. All JAIVAC-1 subjects sero-converted for PfF2 but elicited poor immune response to PfMSP-119. Dose-response relationship was observed between vaccine dose of PfF2 and antibody response. The antibodies against PfF2 were predominantly of IgG1 and IgG3 isotype. Sera from JAIVAC-1 subjects reacted with late schizonts in a punctate pattern in immunofluorescence assays. Purified IgG from JAIVAC-1 sera displayed significant growth inhibitory activity against Plasmodium falciparum CAMP strain. Conclusion Antigen PfF2 should be retained as a component of a recombinant malaria vaccine but PfMSP-119 construct needs to be optimised to improve its immunogenicity. Trial Registration Clinical Trial Registry, India CTRI/2010/091/000301

Production and preclinical evaluation of Plasmodium falciparum MSP-119 and MSP-311 chimeric protein, PfMSP-Fu24.

ABSTRACT A Plasmodium falciparum chimeric protein, PfMSP-Fu24, was constructed by genetically coupling immunodominant, conserved regions of two merozoite surface proteins, the 19-kDa region C-terminal region of merozoite surface protein 1 (PfMSP-119) and an 11-kDa conserved region of merozoite surface protein 3 (PfMSP-311), to augment the immunogenicity potential of these blood-stage malaria vaccine candidates. Here we describe an improved, efficient, and scalable process to produce high-quality PfMSP-Fu24. The chimeric protein was produced in Escherichia coli SHuffle T7 Express lysY cells that express disulfide isomerase DsbC. A two-step purification process comprising metal affinity followed by cation exchange chromatography was developed, and we were able to obtain PfMSP-Fu24 with purity above 99% and with a considerable yield of 23 mg/liter. Immunogenicity of PfMSP-Fu24 formulated with several adjuvants, including Adjuplex, Alhydrogel, Adjuphos, Alhydrogel plus glucopyranosyl lipid adjuvant, aqueous (GLA-AF), Adjuphos+GLA-AF, glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE), and Freunds adjuvant, was evaluated. PfMSP-Fu24 formulated with GLA-SE and Freunds adjuvant in mice and with Alhydrogel and Freunds adjuvant in rabbits produced high titers of PfMSP-119 and PfMSP-311-specific functional antibodies. Some of the adjuvant formulations induced inhibitory antibody responses and inhibited in vitro growth of P. falciparum parasites in the presence as well as in the absence of human monocytes. These results suggest that PfMSP-Fu24 can form a constituent of a multistage malaria vaccine.

Field evaluation of a blood based test for active tuberculosis in endemic settings

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30–70%, and 93–99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.

Antigenicity of a Bacterially Expressed Triple Chimeric Antigen of Plasmodium falciparum AARP, MSP-311 and MSP-119: PfAMSP-Fu35

Development of fusion chimeras as potential vaccine candidates is considered as an attractive strategy to generate effective immune responses to more than one antigen using a single construct. Here, we described the design, production, purification and antigenicity of a fusion chimera (PfAMSP-Fu35), comprised of immunologically relevant regions of three vaccine target malaria antigens, PfAARP, PfMSP-3 and PfMSP-1. The recombinant PfAMSP-Fu35 is expressed as a soluble protein and purified to homogeneity with ease at a yield of ~ 7 mg L-1. Conformational integrity of the C-terminal fragment of PfMSP-1, PfMSP-119 was retained in the fusion chimera as shown by ELISA with conformation sensitive monoclonal antibodies. High titre antibodies were raised to the fusion protein and to all the three individual components in mice and rabbits upon immunization with fusion chimera in two different adjuvant formulations. The sera against PfAMSP-Fu35 recognized native parasite proteins corresponding to the three components of the fusion chimera. As shown by invasion inhibition assay and antibody mediated cellular inhibition assay, antibodies purified from the PfAMSP-Fu35 immunized serum successfully and efficiently inhibited parasite invasion in P. falciparum 3D7 in vitro both directly and in monocyte dependent manner. However, the invasion inhibitory activity of anti-AMSP-Fu35 antibody is not significantly enhanced as expected as compared to a previously described two component fusion chimera, MSP-Fu24. Therefore, it may not be of much merit to consider AMSP-Fu35 as a vaccine candidate for preclinical development.

Protein–protein interaction studies reveal the Plasmodium falciparum merozoite surface protein-1 region involved in a complex formation that binds to human erythrocytes

Plasmodium falciparum merozoite surface protein (PfMSP) 1 has been studied extensively as a vaccine candidate antigen. PfMSP-1 undergoes proteolytic processing into four major products, such as p83, p30, p38, and p42, that are associated in the form of non-covalent complex(s) with other MSPs. To delineate MSP1 regions involved in the interaction with other MSPs, here we expressed recombinant proteins (PfMSP-165) encompassing part of p38 and p42 regions and PfMSP-119 PfMSP-165 interacted strongly with PfMSP-3, PfMSP-6, PfMSP-7, and PfMSP-9, whereas PfMSP-119 did not interact with any of these proteins. Since MSP-1 complex binds human erythrocytes, we examined the ability of these proteins to bind human erythrocyte. Among the proteins of MSP-1 complex, PfMSP-6 and PfMSP-9 bound to human erythrocytes. Serological studies showed that PfMSP-165 was frequently recognized by sera from malaria endemic regions, whereas this was not the case for PfMSP-119 In contrast, antibodies against PfMSP-119 showed much higher inhibition of merozoite invasion compared with antibodies against the larger PfMSP-165 fragment. Importantly, anti-PfMSP-119 antibodies recognized both recombinant proteins, PfMSP-119 and PfMSP-165; however, anti-PfMSP-165 antibody failed to recognize the PfMSP-119 protein. Taken together, these results demonstrate that PfMSP-1 sequences upstream of the 19 kDa C-terminal region are involved in molecular interactions with other MSPs, and these sequences may probably serve as a smoke screen to evade antibody response to the membrane-bound C-terminal 19 kDa region.