Natural infections with different Plasmodium species induce antibodies reactive to a chimeric Plasmodium vivax recombinant protein.

BACKGROUND: As malaria incidence and transmission in a region decreases, it becomes increasingly difficult to identify areas of active transmission. Improved methods for identifying and monitoring foci of active malaria transmission are needed in areas of low parasite prevalence in order to achieve malaria elimination. Serological assays can provide population-level infection history to inform elimination campaigns. METHODS: A bead-based multiplex antibody detection assay was used to evaluate a chimeric Plasmodium vivax MSP1 protein (PvRMC-MSP1), designed to be broadly immunogenic for use in vaccine studies, to act as a pan-malaria serological tool based on its ability to capture IgG in plasma samples obtained from naturally exposed individuals. Samples from 236 US travellers with PCR confirmed infection status from all four major Plasmodium species infecting humans, Plasmodium falciparum (n = 181), Plasmodium vivax (n = 38), Plasmodium malariae (n = 4), and Plasmodium ovale (n = 13) were tested for IgG capture using PvRMC-MSP1 as well as the four recombinant MSP1-19 kD isoforms representative of these Plasmodium species. RESULTS: Regardless of infecting Plasmodium species, a large proportion of plasma samples from infected US travellers provided a high assay signal to the PvRMC-MSP1 chimeric protein, with 115 high responders out of 236 samples assessed (48.7%). When grouped by active infection, 38.7% P. falciparum-, 92.1% of P. vivax-, 75.0% P. malariae-, and 53.4% of P. ovale-infected individuals displayed high assay signals in response to PvRMC-MSP1. It was also determined that plasma from P. vivax-infected individuals produced increased assay signals in response to the PvRMC-MSP1 chimera as compared to the recombinant PvMSP1 for 89.5% (34 out of 38) of individuals. PvRMC-MSP1 also showed improved ability to capture IgG antibodies from P. falciparum-infected individuals when compared to the capture by recombinant PvMSP1, with high assay signals observed for 38.7% of P. falciparum-infected travellers in response to PvRMC-MSP1 IgG capture compared to just 1.1% who were high responders to capture by the recombinant PvMSP1 protein. CONCLUSIONS: These results support further study of designed antigens as an approach for increasing sensitivity or broadening binding capacity to improve existing serological tools for determining population-level exposure to Plasmodium species. Including both broad-reacting and Plasmodium species-specific antigen-coated beads in an assay panel could provide a nuanced view of population-level exposure histories, an extensive IgG profile, and detailed seroestimates. A more sensitive serological tool for detection of P. vivax exposure would aid malaria elimination campaigns in co-endemic areas and regions where P. vivax is the dominant parasite.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.

BACKGROUND: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. METHODS: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. RESULTS: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. CONCLUSIONS: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

The endoplasmic reticulum chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterised ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localises to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

A Recombinant Chimeric Ad5/3 Vector Expressing a Multistage Plasmodium Antigen Induces Protective Immunity in Mice Using Heterologous Prime-Boost Immunization Regimens.

An ideal malaria vaccine should target several stages of the parasite life cycle and induce antiparasite and antidisease immunity. We have reported a Plasmodium yoelii chimeric multistage recombinant protein (P. yoelii linear peptide chimera/recombinant modular chimera), engineered to express several autologous T cell epitopes and sequences derived from the circumsporozoite protein and the merozoite surface protein 1. This chimeric protein elicits protective immunity, mediated by CD4(+) T cells and neutralizing Abs. However, experimental evidence, from pre-erythrocytic vaccine candidates and irradiated sporozoites, has shown that CD8(+) T cells play a significant role in protection. Recombinant viral vectors have been used as a vaccine platform to elicit effective CD8(+) T cell responses. The human adenovirus (Ad) serotype 5 has been tested in malaria vaccine clinical trials with excellent safety profile. Nevertheless, a major concern for the use of Ad5 is the high prevalence of anti-vector neutralizing Abs in humans, hampering its immunogenicity. To minimize the impact of anti-vector pre-existing immunity, we developed a chimeric Ad5/3 vector in which the knob region of Ad5 was replaced with that of Ad3, conferring partial resistance to anti-Ad5 neutralizing Abs. Furthermore, we implemented heterologous Ad/protein immunization regimens that include a single immunization with recombinant Ad vectors. Our data show that immunization with the recombinant Ad5/3 vector induces protective efficacy indistinguishable from that elicited by Ad5. Our study also demonstrates that the dose of the Ad vectors has an impact on the memory profile and protective efficacy. The results support further studies with Ad5/3 for malaria vaccine development.

Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera.

Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4(+) and CD8(+) PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development.

A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by three-dimensional tomography in the caveola-vesicle complexes (Schüffner's dots) of infected erythrocytes is a member of the PHIST family.

Plasmodium vivax and P. cynomolgi produce numerous caveola-vesicle complex (CVC) structures within the surface of the infected erythrocyte membrane. These contrast with the electron-dense knob protrusions expressed at the surface of Plasmodium falciparum-infected erythrocytes. Here we investigate the three-dimensional (3-D) structure of the CVCs and the identity of a predominantly expressed 95 kDa CVC protein. Liquid chromatography - tandem mass spectrometry analysis of immunoprecipitates by monoclonal antibodies from P. cynomolgi extracts identified this protein as a member of the Plasmodium helical interspersed subtelomeric (PHIST) superfamily with a calculated mass of 81 kDa. We named the orthologous proteins PvPHIST/CVC-81(95) and PcyPHIST/CVC-81(95) , analysed their structural features, including a PEXEL motif, repeated sequences and a C-terminal PHIST domain, and show that PHIST/CVC-81(95) is most highly expressed in trophozoites. We generated images of CVCs in 3-D using electron tomography (ET), and used immuno-ET to show PHIST/CVC-81(95) localizes to the cytoplasmic side of the CVC tubular extensions. Targeted gene disruptions were attempted in vivo. The pcyphist/cvc-81(95) gene was not disrupted, but parasites containing episomes with the tgdhfr selection cassette were retrieved by selection with pyrimethamine. This suggests that PHIST/CVC-81(95) is essential for survival of these malaria parasites.

A hybrid multistage protein vaccine induces protective immunity against murine malaria.

We have previously reported the design and expression of chimeric recombinant proteins as an effective platform to deliver malaria vaccines. The erythrocytic and exoerythrocytic protein chimeras described included autologous T helper epitopes genetically linked to defined B cell epitopes. Proof-of-principle studies using vaccine constructs based on the Plasmodium yoelii circumsporozoite protein (CSP) and P. yoelii merozoite surface protein-1 (MSP-1) showed encouraging results when tested individually in this mouse malaria model. To evaluate the potential synergistic or additive effect of combining these chimeric antigens, we constructed a synthetic gene encoding a hybrid protein that combined both polypeptides in a single immunogen. The multistage vaccine was expressed in soluble form in Escherichia coli at high yield. Here we report that the multistage protein induced robust immune responses to individual components, with no evidence of vaccine interference. Passive immunization using purified IgG from rabbits immunized with the hybrid protein conferred more robust protection against the experimental challenge with P. yoelii sporozoites than passive immunization with purified IgG from rabbits immunized with the individual proteins. High antibody titers and high frequencies of CD4(+)- and CD8(+)-specific cytokine-secreting T cells were elicited by vaccination. T cells were multifunctional and able to simultaneously produce interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). The mechanism of vaccine-induced protection involved neutralizing antibodies and effector CD4(+) T cells and resulted in the control of hyperparasitemia and protection against malarial anemia. These data support our strategy of using an array of autologous T helper epitopes to maximize the response to multistage malaria vaccines.

Cannabinoid receptor-2 (CB2) agonist ameliorates colitis in IL-10(-/-) mice by attenuating the activation of T cells and promoting their apoptosis.

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptor induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10(-/-) mice. JWH-133 effectively attenuated the overall clinical score, and reversed colitis-associated pathogenesis and decrease in body weight in IL-10(-/-) mice. After JWH-133 treatment, the percentage of CD4(+) T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells declined in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN) of mice with chronic colitis. JWH-133 was also effective in ameliorating dextran sodium sulfate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodo-pravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, and suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD.

Role of resveratrol-induced CD11b(+) Gr-1(+) myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3(+) T cells and amelioration of chronic colitis in IL-10(-/-) mice.

Resveratrol, a naturally occurring polyphenol has received significant attention as a potent anti-inflammatory agent. Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce proinflammatory cytokines. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population characterized by the co-expression of CD11b(+) and Gr-1(+) and have long been known for their immunosuppressive function. We report that resveratrol effectively attenuated overall clinical scores as well as various pathological markers of colitis in IL-10(-/-) mice by down regulating Th1 responses. Resveratrol lessened the colitis-associated decrease in body weight and increased levels of serum amyloid A (SAA), CXCL10 and colon TNF-α, IL-6, RANTES, IL-12 and IL-1ß concentrations. After resveratrol treatment, the percentage of CXCR3 expressing T cells was decreased in the spleen, mesenteric lymph nodes (MLN), and intestinal lamina propria (LP). However, the percentage and absolute numbers of CD11b(+) and Gr-1(+)cells in the lamina propria (LP) and spleen were increased after resveratrol treatment as compared with the vehicle treatment. Co-culture of resveratrol-induced CD11b(+) Gr-1(+) cells with T cells, attenuated T cell proliferation, and most importantly reduced IFN-γ and GM-CSF production by LP derived T cells from vehicle treated IL-10(-/-) mice with chronic colitis. The current study suggests that administration of resveratrol into IL-10(-/-) mice induces immunosuppressive CD11b(+) Gr-1(+) MDSCs in the colon, which correlates with reversal of established chronic colitis, and down regulation of mucosal and systemic CXCR3(+) expressing effector T cells as well as inflammatory cytokines in the colon. The induction of immunosuppressive CD11b(+) Gr-1(+) cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol.

The use of a chimeric antigen for Plasmodium falciparum and P. vivax seroprevalence estimates from community surveys in Ethiopia and Costa Rica.

BACKGROUND: In low-transmission settings, accurate estimates of malaria transmission are needed to inform elimination targets. Detection of antimalarial antibodies provides exposure history, but previous studies have mainly relied on species-specific antigens. The use of chimeric antigens that include epitopes from multiple species of malaria parasites in population-based serological surveys could provide data for exposure to multiple Plasmodium species circulating in an area. Here, the utility of P. vivax/P. falciparum chimeric antigen for assessing serological responses was evaluated in Ethiopia, an endemic country for all four human malarias, and Costa Rica, where P. falciparum has been eliminated with reports of sporadic P. vivax cases. METHODS: A multiplex bead-based assay was used to determine the seroprevalence of IgG antibodies against a chimeric malaria antigen (PvRMC-MSP1) from blood samples collected from household surveys in Ethiopia in 2015 (n = 7,077) and Costa Rica in 2015 (n = 851). Targets specific for P. falciparum (PfMSP1) and P. vivax (PvMSP1) were also included in the serological panel. Seroprevalence in the population and seroconversion rates were compared among the three IgG targets. RESULTS: Seroprevalence in Costa Rica was 3.6% for PfMSP1, 41.5% for PvMSP1 and 46.7% for PvRMC-MSP1. In Ethiopia, seroprevalence was 27.6% for PfMSP1, 21.4% for PvMSP1, and 32.6% for PvRMC-MSP1. IgG levels in seropositive individuals were consistently higher for PvRMC-MSP1 when compared to PvMSP1 in both studies. Seroconversion rates were 0.023 for PvMSP1 and 0.03 for PvRMC-MSP1 in Costa Rica. In Ethiopia, seroconversion rates were 0.050 for PfMSP1, 0.044 for PvMSP1 and 0.106 for PvRMC-MSP1. CONCLUSIONS: Our data indicate that chimeric antigen PvRMC-MSP1 is able to capture antibodies to multiple epitopes from both prior P. falciparum and P. vivax infections, and suitable chimeric antigens can be considered for use in serosurveys with appropriate validation.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018.

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Resveratrol (trans-3,5,4'-trihydroxystilbene) induces silent mating type information regulation-1 and down-regulates nuclear transcription factor-kappaB activation to abrogate dextran sulfate sodium-induced colitis.

Inflammatory bowel disease is a chronic, relapsing, and tissue-destructive disease. Resveratrol (3,4,5-trihydroxy-trans-stilbene), a naturally occurring polyphenol that exhibits beneficial pleiotropic health effects, is recognized as one of the most promising natural molecules in the prevention and treatment of chronic inflammatory disease and autoimmune disorders. In the present study, we investigated the effect of resveratrol on dextran sodium sulfate (DSS)-induced colitis in mice and found that it effectively attenuated overall clinical scores as well as various pathological markers of colitis. Resveratrol reversed the colitis-associated decrease in body weight and increased levels of serum amyloid A, tumor necrosis factor-alpha, interleukin (IL-6), and IL-1beta. After resveratrol treatment, the percentage of CD4(+) T cells in mesenteric lymph nodes (MLN) of colitis mice was restored to normal levels, and there was a decrease in these cells in the colon lamina propria (LP). Likewise, the percentages of macrophages in MLN and the LP of mice with colitis were decreased after resveratrol treatment. Resveratrol also suppressed cyclooxygenase-2 (COX-2) expression induced in DSS-exposed mice. Colitis was associated with a decrease in silent mating type information regulation-1 (SIRT1) gene expression and an increase in p-inhibitory kappaB expression and nuclear transcription factor-kappaB (NF-kappaB) activation. Resveratrol treatment of mice with colitis significantly reversed these changes. This study demonstrates for the first time that SIRT1 is involved in colitis, functioning as an inverse regulator of NF-kappaB activation and inflammation. Furthermore, our results indicate that resveratrol may protect against colitis through up-regulation of SIRT1 in immune cells in the colon.

A Multi-Stage Plasmodium vivax Malaria Vaccine Candidate Able to Induce Long-Lived Antibody Responses Against Blood Stage Parasites and Robust Transmission-Blocking Activity.

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Treatment of Tympanic Membrane Retraction Pockets by Excision and Cartilage Tympanoplasty: A Prospective Study.

To assess the role of cartilage tympanoplasty in management of retraction pockets of the pars flaccida. This was a prospective study at a tertiary care centre. Twenty patients having grade III or grade IV retraction pockets were included in the study. Retraction pockets were treated by excision and cartilage tympanoplasty. Findings noted on follow-up were recorded and analysed. Graft was taken up in 18 (90%) cases with residual perforation in 2 (10%) cases. Recurrence of retraction pockets was observed in 6 (30%) cases. Hearing was improved up to 15 dB in 16 (80%) cases. It is concluded that grade III and IV retraction pockets can be well managed by excision and cartilage tympanoplasty.

TACI Contributes to Plasmodium yoelii Host Resistance by Controlling T Follicular Helper Cell Response and Germinal Center Formation.

The delay in parasite-specific B cell development leaves people in malaria endemic areas vulnerable to repeated Plasmodium infections. Here, we investigated the role of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), a molecule involved in the generation of antigen-specific antibody secreting cells, in host response to non-lethal Plasmodium yoelii infection. We found that TACI deficiency not only resulted in higher peak parasitemia levels in P. yoelii challenged mice, but also led to a delay in parasite clearance and anti-P. yoelii Merozoite Surface Protein 1(C-terminal 19-kDa fragment [rMSP-119]) protein and anti-rMSP-119 and anti-P. yoelii IgG antibody development. There was also a delay in the generation of splenic high affinity antibody secreting cells that recognize rMSP-119 protein as compared to wild-type mice. Interestingly, coinciding with the delay in parasite clearance there was a delay in the resolution of T follicular helper (TFH) cell and germinal center (GC) B cell responses in TACI -/- mice. The persistence of TFH and GC B cells is likely a result of enhanced interaction between TFH and GC B cells because inducible costimulator ligand (ICOSL) expression was significantly higher on TACI -/- GC B cells than wild-type cells. The difference in the kinetics of GC reaction appeared to also impact the emergence of plasma cells (PC) because there was a delay in the generation of TACI -/- mice PC. Nevertheless, following the recovery from P. yoelii infection, TACI -/- and wild-type mice were both protected from a rechallenge infection. Establishment of protective B cell response was responsible for the resolution of parasitemia because B cells purified from recovered TACI -/- or wild-type mice were equally protective when introduced to naïve wild-type mice prior to P. yoelii challenge. Thus, despite the increased susceptibility of TACI -/- mice to P. yoelii infection and a delay in the development of protective antibody levels, TACI -/- mice are able to clear the infection and resist rechallenge infection.

Inclusion of the murine IgGκ signal peptide increases the cellular immunogenicity of a simian adenoviral vectored Plasmodium vivax multistage vaccine.

INTRODUCTION: Cellular and humoral immune responses are both involved in protection against Plasmodium infections. The only malaria vaccine available, RTS,S, primarily induces short-lived antibodies and targets only a pre-erythrocytic stage antigen. Inclusion of erythrocytic stage targets and enhancing cellular immunogenicity are likely necessary for developing an effective second-generation malaria vaccine. Adenovirus vectors have been used to improve the immunogenicity of protein-based vaccines. However, the clinical assessment of adenoviral-vectored malaria vaccines candidates has shown the induction of robust Plasmodium-specific CD8+ but not CD4+ T cells. Signal peptides (SP) have been used to enhance the immunogenicity of DNA vaccines, but have not been tested in viral vector vaccine platforms. OBJECTIVES: The objective of this study was to determine if the addition of the SP derived from the murine IgGκ light chain within a recombinant adenovirus vector encoding a multistage P. vivax vaccine candidate could improve the CD4+ T cell response. METHODS: In this proof-of-concept study, we immunized CB6F1/J mice with either the recombinant simian adenovirus 36 vector containing the SP (SP-SAd36) upstream from a transgene encoding a chimeric P. vivax multistage protein or the same SAd36 vector without the SP. Mice were subsequently boosted twice with the corresponding recombinant proteins emulsified in Montanide ISA 51 VG. Immunogenicity was assessed by measurement of antibody quantity and quality, and cytokine production by T cells after the final immunization. RESULTS: The SP-SAd36 immunization regimen induced significantly higher antibody avidity against the chimeric P. vivax proteins tested and higher frequencies of IFN-γ and IL-2 CD4+ and CD8+ secreting T cells, when compared to the unmodified SAd36 vector. CONCLUSIONS: The addition of the murine IgGκ signal peptide significantly enhances the immunogenicity of a SAd36 vectored P. vivax multi-stage vaccine candidate in mice. The potential of this approach to improve upon existing viral vector vaccine platforms warrants further investigation.

Nontuberculous mycobacterial infections in Indian AIDS patients detected by a novel set of ESAT-6 polymerase chain reaction primers.

Nontuberculous mycobacteria are often underdiagnosed due to lack of proper diagnostic facilities. To overcome this, we created a rapid PCR method for the species-specific diagnosis of Mycobacterium tuberculosis and its differentiation from other mycobacteria. A set of PCR primers targeting the gene encoding for early-secreted antigen-6 (ESAT-6) of the M. tuberculosis complex was designed and standardized on mycobacterial standard strains and on 75 recent isolates from AIDS patients and 70 isolates from HIV-negative patients seen at the hospital of the All India Institute of Medical Sciences, New Delhi, India. All 145 fresh mycobacterial isolates were identified using phenotypic methods and 16S rRNA PCR followed by sequencing of hypervariable region A. The ESAT-6 PCR detected all of the M. tuberculosis strains correctly (100% sensitivity), but none of the nontuberculous Mycobacterium spp. gave positive results (100% specific). Most nontuberculous mycobacteria were identified in patients with AIDS (24%) followed by those with tuberculous lymphadenitis (12.5%) and those with pulmonary tuberculosis whose treatment had failed (4.3%). The most common nontuberculous mycobacterial species isolated from AIDS patients was M. avium (6.6%), followed by M. fortuitum (5.7%), M. intracellulare and M. terrae (2.6% each). M. celatum, M. duvalii, M. austroafricanum, M. phlei and M. flavescence were also isolated from one patient each. The combination of genus-specific PCR primers with the novel ESAT-6 primer set could provide accurate and rapid diagnosis of mycobacteriosis.

A prime-boost immunization regimen based on a simian adenovirus 36 vectored multi-stage malaria vaccine induces protective immunity in mice.

Malaria remains a considerable burden on public health. In 2015, the WHO estimates there were 212 million malaria cases causing nearly 429,000 deaths globally. A highly effective malaria vaccine is needed to reduce the burden of this disease. We have developed an experimental vaccine candidate (PyCMP) based on pre-erythrocytic (CSP) and erythrocytic (MSP1) stage antigens derived from the rodent malaria parasite P. yoelii. Our protein-based vaccine construct induces protective antibodies and CD4+ T cell responses. Based on evidence that viral vectors increase CD8+ T cell-mediated immunity, we also have tested heterologous prime-boost immunization regimens that included human adenovirus serotype 5 vector (Ad5), obtaining protective CD8+ T cell responses. While Ad5 is commonly used for vaccine studies, the high prevalence of pre-existing immunity to Ad5 severely compromises its utility. Here, we report the use of the novel simian adenovirus 36 (SAd36) as a candidate for a vectored malaria vaccine since this virus is not known to infect humans, and it is not neutralized by anti-Ad5 antibodies. Our study shows that the recombinant SAd36PyCMP can enhance specific CD8+ T cell response and elicit similar antibody titers when compared to an immunization regimen including the recombinant Ad5PyCMP. The robust immune responses induced by SAd36PyCMP are translated into a lower parasite load following P. yoelii infectious challenge when compared to mice immunized with Ad5PyCMP.

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens.

Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.