Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine.

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230-Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.

Peripheral Merozoite Surface Proteins Are Targets of Naturally Acquired Immunity against Malaria in both India and Ghana.

Development of a successful blood-stage vaccine against Plasmodium falciparum malaria remains a high priority. Immune-epidemiological studies are effective tools for the identification of antigenic targets of naturally acquired immunity (NAI) against malaria. However, differences in study design and methodology may compromise interstudy comparisons. Here, we assessed antibody responses against intact merozoites and a panel of 24 recombinant merozoite antigens in longitudinal cohort studies of Ghanaian (n = 115) and Indian (n = 121) populations using the same reagents and statistical methods. Anti-merozoite antibodies were associated with NAI in both the Indian (hazard ratio [HR] = 0.41, P = 0.020) and the Ghanaian (HR = 0.17, P < 0.001) participants. Of the 24 antigen-specific antibodies quantified, 12 and 8 were found to be protective in India and Ghana, respectively. Using least absolute shrinkage and selection operator (LASSO) regression, a powerful variable subselection technique, we identified subsets of four (MSP6, MSP3.7, MSPDBL2, and Pf12) and five (cMSP33D7, MSP3.3, MSPDBL1, GLURP-R2, and RALP-1) antigens that explained NAI better than the individual antibodies in India (HR = 0.18, P < 0.001) and Ghana (HR = 0.31, P < 0.001), respectively. IgG1 and/or IgG3 subclasses against five antigens from these subsets were associated with protection. Through this comparative study, maintaining uniformity of reagents and methodology, we demonstrate that NAI across diverse geographic regions may result from antibodies to multiple antigenic targets that constitute the peripheral merozoite surface protein complexes.

A Reproducible and Scalable Process for Manufacturing a Pfs48/45 Based Plasmodium falciparum Transmission-Blocking Vaccine.

The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb45.1. Intact mass analysis of R0.6C confirmed the identity of the product including the three disulfide bonds and the absence of post-translational modifications. Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography (SEC-MALS), further confirmed that R0.6C was monomeric (~70 kDa) in solution. Lastly, preclinical studies demonstrated that the R0.6C Drug Product (adsorbed to Alhydrogel®) elicited functional antibodies in small rodents and that adding Matrix-M™ adjuvant further increased the functional response. Here, building upon our past work, we filled the gap between laboratory and manufacturing to ready R0.6C for production under cGMP and eventual clinical evaluation as a malaria TB vaccine.

The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable.

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis-derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

Plasmodium falciparum Clag9-Associated PfRhopH Complex Is Involved in Merozoite Binding to Human Erythrocytes.

Cytoadherence-linked asexual gene 9 (Clag9), a conserved Plasmodium protein expressed during the asexual blood stages, is involved in the cytoadherence of infected red blood cells (RBCs) to the endothelial lining of blood vessels. Here, we show that Plasmodium falciparum Clag9 (PfClag9) is a component of the PfClag9-RhopH complex that is involved in merozoite binding to human erythrocytes. To characterize PfClag9, we expressed four fragments of PfClag9, encompassing the entire protein. Immunostaining analysis using anti-PfClag9 antibodies showed expression and localization of PfClag9 at the apical end of the merozoites. Mass spectrometric analysis of merozoite extracts after immunoprecipitation using anti-PfClag9 antibody identified P. falciparum rhoptry-associated protein 1 (PfRAP1), PfRAP2, PfRAP3, PfRhopH2, and PfRhopH3 as associated proteins. The identified rhoptry proteins were expressed, and their association with PfClag9 domains was assessed by using protein-protein interaction tools. We further showed that PfClag9 binds human RBCs by interacting with the glycophorin A-band 3 receptor-coreceptor complex. In agreement with its cellular localization, PfClag9 was strongly recognized by antibodies generated during natural infection. Mice immunized with the C-terminal domain of PfClag9 were partially protected against a subsequent challenge infection with Plasmodium berghei, further supporting a biological role of PfClag9 during natural infection. Taken together, these results provide direct evidence for the existence of a PfRhopH-Clag9 complex on the Plasmodium merozoite surface that binds to human RBCs.

Pfs230 and Pfs48/45 Fusion Proteins Elicit Strong Transmission-Blocking Antibody Responses Against Plasmodium falciparum.

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are expressed during transmission from man to mosquito and are leading candidates for a malaria transmission blocking vaccine. Individually they generate transmission blocking (TB) antibodies in rodent models. Whether the single protein vaccines are suitable to use in field settings will primarily depend on their potency to elicit functional antibodies. We hypothesized that a combination of both proteins will be more potent than each protein individually. Therefore we designed chimeric proteins composed of fragments of both Pfs230 and Pfs48/45 as well as single protein fragments, and expressed these in Lactoccus lactis. Both the individual Pfs230 and Pfs48/45 fragments and chimeras elicited high levels of functional antibodies in mice. Importantly, one of the chimeric proteins elicited over threefold higher transmission blocking antibody responses than the single antigens alone. Furthermore the immunogenicity of one of the chimeras could be enhanced through coupling to a virus-like particle (VLP). Altogether these data support further clinical development of these novel constructs.

Biochemical characterization of Plasmodium complement factors binding protein for its role in immune modulation.

Complement system is the first line of human defence against intruding pathogens and is recognized as a potentially useful therapeutic target. Human malaria parasite Plasmodium employs a series of intricate mechanisms that enables it to evade different arms of immune system, including the complement system. Here, we show the expression of a multi-domain Plasmodium Complement Control Protein 1, PfCCp1 at asexual blood stages and its binding affinity with C3b as well as C4b proteins of human complement cascade. Using a biochemical assay, we demonstrate that PfCCp1 binds with complement factors and inhibits complement activation. Active immunization of mice with PfCCp1 followed by challenge with Plasmodium berghei resulted in the loss of biphasic growth of parasites and early death in comparison to the control group. The study also showed a role of PfCCp1 in modulating Toll-like receptor (TLR)-mediated signalling and effector responses on antigen-presenting cells. PfCCp1 binds with dendritic cells that down-regulates the expression of signalling molecules and pro-inflammatory cytokines, thereby dampening the TLR2-mediated signalling; hence acting as a potent immuno-modulator. In summary, PfCCp1 appears to be an important component of malaria parasite directed immuno-modulating strategies that promote the adaptive fitness of pathogens in the host.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Cytophilic Antibodies Against Key Plasmodium falciparum Blood Stage Antigens Contribute to Protection Against Clinical Malaria in a High Transmission Region of Eastern India.

Background: The collection of clinical data from a tribal population in a malaria-endemic area of India suggests the occurrence of naturally acquired immunity (NAI) against Plasmodium falciparum malaria. Methods: Quantity and functionality of immunoglobulin G (IgG) antibodies against intact merozoites and recombinant proteins were assessed in a 13-month longitudinal cohort study of 121 individuals, 3-60 years of age. Results: Opsonic phagocytosis of merozoites activity was strongly associated (hazard ratio [HR] = 0.34; 95% confidence interval [CI] = .18-.66; P = .0013) with protection against febrile malaria. Of the different IgG subclasses, only IgG3 antibodies against intact whole merozoites was significantly associated with protection against febrile malaria (HR = 0.47; 95% CI = .26-.86; P = .01). Furthermore, a combination of IgG3 antibody responses against Pf12, MSP3.7, MSP3.3, and MSP2FC27 was strongly associated with protection against febrile malaria (HR = 0.15; 95% CI, .06-.37; P = .0001). Conclusions: These data suggest that NAI may, at least in part, be explained by opsonic phagocytosis of merozoites and IgG3 responses against whole merozoites, and in particular to a combination of 4 antigens is critical in this population. These results may have implications in the development of a subunit malaria vaccine. Opsonic phagocytosis of Plasmodium falciparum merozoites was associated with protection against clinical malaria in an India population. Antibody profiling identified four merozoite antigens (Pf12, MSP3.7, MSP3.3, and MSP2) as targets of protective Immunoglobuline G3 antibodies.

Lactococcus lactis provides an efficient platform for production of disulfide-rich recombinant proteins from Plasmodium falciparum.

BACKGROUND: The production of recombinant proteins with proper conformation, appropriate post-translational modifications in an easily scalable and cost-effective system is challenging. Lactococcus lactis has recently been identified as an efficient Gram positive cell factory for the production of recombinant protein. We and others have used this expression host for the production of selected malaria vaccine candidates. The safety of this production system has been confirmed in multiple clinical trials. Here we have explored L. lactis cell factories for the production of 31 representative Plasmodium falciparum antigens with varying sizes (ranging from 9 to 90 kDa) and varying degree of predicted structural complexities including eleven antigens with multiple predicted structural disulfide bonds, those which are considered difficult-to-produce proteins. RESULTS: Of the 31 recombinant constructs attempted in the L. lactis expression system, the initial expression efficiency was 55% with 17 out of 31 recombinant gene constructs producing high levels of secreted recombinant protein. The majority of the constructs which failed to produce a recombinant protein were found to consist of multiple intra-molecular disulfide-bonds. We found that these disulfide-rich constructs could be produced in high yields when genetically fused to an intrinsically disorder protein domain (GLURP-R0). By exploiting the distinct biophysical and structural properties of the intrinsically disordered protein region we developed a simple heat-based strategy for fast purification of the disulfide-rich protein domains in yields ranging from 1 to 40 mg/l. CONCLUSIONS: A novel procedure for the production and purification of disulfide-rich recombinant proteins in L. lactis is described.

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.

Human Cyclophilin B forms part of a multi-protein complex during erythrocyte invasion by Plasmodium falciparum.

Invasion of human erythrocytes by Plasmodium falciparum merozoites involves multiple interactions between host receptors and their merozoite ligands. Here we report human Cyclophilin B as a receptor for PfRhopH3 during merozoite invasion. Localization and binding studies show that Cyclophilin B is present on the erythrocytes and binds strongly to merozoites. We demonstrate that PfRhopH3 binds to the RBCs and their treatment with Cyclosporin A prevents merozoite invasion. We also show a multi-protein complex involving Cyclophilin B and Basigin, as well as PfRhopH3 and PfRh5 that aids the invasion. Furthermore, we report identification of a de novo peptide CDP3 that binds Cyclophilin B and blocks invasion by up to 80%. Collectively, our data provide evidence of compounded interactions between host receptors and merozoite surface proteins and paves the way for developing peptide and small-molecules that inhibit the protein-protein interactions, individually or in toto, leading to abrogation of the invasion process.

Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine.

BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. RESULTS: A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats. CONCLUSIONS: The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.

Varied presentations of leptospirosis: experience from a tertiary care hospital in north India.

Leptospirosis has been recognised as an emerging global public health problem. The aim of our study was to explore the epidemiological and clinical pattern of disease occurrence in suspected cases and to search for any existing co-infections. Ours was a retrospective study in patients with acute febrile illness in north India over a period of three years (April 2011 to June 2014). Serological diagnosis of leptospirosis was made using the PanBio IgM ELISA kit. Using modified Faine's criteria, presumptive and possible diagnosis was made in 57% and 34% cases, respectively. Most of the affected population was resident in north and central India. Nineteen patients showed co-infection with other common pathogens prevailing locally. There is a need to increase awareness and understand the local sero-epidemiological pattern of leptospirosis so that timely preventive and curative action may be taken by healthcare authorities.

Delineation of immunodominant and cytadherence segment(s) of Mycoplasma pneumoniae P1 gene.

BACKGROUND: Adhesion of Mycoplasma pneumoniae (M. pneumoniae) to host epithelial cells requires several adhesin proteins like P1, P30 and P116. Among these proteins, P1 protein has been inedited as one of the major adhesin and immunogenic protein present on the attachment organelle of M. pneumoniae. In the present study, we scanned the entire sequence of M. pneumoniae P1 protein to identify the immunodominant and cytadherence region(s). M. pneumoniae P1 gene was synthesized in four segments replacing all the UGA codons to UGG codons. Each of the four purified P1 protein fragment was analyzed for its immunogenicity with anti-M. pneumoniae M129 antibodies (Pab M129) and sera of M. pneumoniae infected patients by western blotting and ELISA. Antibodies were produced against all the P1 protein fragments and these antibodies were used for M. pneumoniae adhesion, M. pneumoniae adhesion inhibition and M. pneumoniae surface exposure assays using HEp-2 cells lines. RESULTS: Our results show that the immunodominant regions are distributed throughout the entire length of P1 protein, while only the N- and C- terminal region(s) of P1 protein are surface exposed and block cytadhesion to HEp-2 cells, while antibodies to two middle fragments failed to block cytadhesion. CONCLUSIONS: These results have important implications in designing strategies to block the attachment of M. pneumoniae to epithelial cells, thus preventing the development of atypical pneumonia.

Identification of an N-terminal 27 kDa fragment of Mycoplasma pneumoniae P116 protein as specific immunogen in M. pneumoniae infections.

BACKGROUND: Mycoplasma pneumoniae is an important cause of respiratory tract infection and is increasingly being associated with other diseases such as asthma and extra-pulmonary complications. Considerable cross-reactivity is known to exist between the whole cell antigens used in the commercial serological testing assays. Identification of specific antigens is important to eliminate the risk of cross-reactions among different related organisms. Adherence of M. pneumoniae to human epithelial cells is mediated through a well defined apical organelle to which a number of proteins such as P1, P30, P116 and HMW1-3 have been localized, and are being investigated for adhesion, gliding and immunodiagnostic purposes. METHODS: A 609 bp fragment P116(N-27), corresponding to the N-terminal region of M. pneumoniae P116 gene was cloned and expressed. A C-terminal fragment P1(C-40), of P1 protein of M. pneumoniae was also expressed. Three IgM ELISA assays based on P116(N-27), P1(C-40) and (P116 (N-27) + P1(C-40)) proteins were optimized and a detailed analysis comparing the reactivity of these proteins with a commercial kit was carried out. Comparative statistical analysis of these assays was performed with the SPSS version 15.0. RESULTS: The expressed P116(N-27) protein was well recognized by the patient sera and was immunogenic in rabbit. P1(C-40) of M. pneumoniae was also immunogenic in rabbit. In comparison to the reference kit, which is reported to be 100% sensitive and 75% specific, ELISA assay based on purified P116(N-27), P1(C-40) and (P116(N-27) + P1(C-40)) proteins showed 90.3%, 87.1% and 96.8% sensitivity and 87.0%, 87.1% and 90.3% specificity respectively. The p value for all the three assays was found to be < 0.001, and there was a good correlation and association between them. CONCLUSION: This study shows that an N-terminal fragment of P116 protein holds a promise for serodiagnosis of M. pneumoniae infection. The IgM ELISA assays based on the recombinant proteins seem to be suitable for the use in serodiagnosis of acute M. pneumoniae infections. The use of short recombinant fragments of P116 and P1 proteins as specific antigens may eliminate the risk of cross-reactions and help to develop a specific and sensitive immunodiagnostic assay for M. pneumoniae detection.