Commensal Neisseria cinerea outer membrane vesicles as a platform for the delivery of meningococcal and gonococcal antigens to the immune system.

An affordable, accessible, and broadly protective vaccine is required to tackle the re-occurring bacterial meningococcal epidemics in Sub-Saharan Africa as well as an effective control of multi-drug resistant strains of gonococcus. Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria represent an attractive platform for antigen delivery to the immune system and therefore for development of multi-component vaccines. In this study, we describe the generation of modified OMVs (mOMVs) from commensal biosafety-level 1 (BSL-1) Neisseria cinerea ATCC® 14685TM, which is phylogenetically close to the pathogenic bacteria Neisseria meningitidis and Neisseria gonorrhoeae. mOMVs were prepared from N. cinerea engineered to express heterologous antigens from N. meningitidis (factor H binding protein (fHbp) and Neisseria Heparin Binding Antigen (NHBA-2)) and from N. gonorrhoeae (NHBA-542). Mice immunised with the mOMVs produced antibodies against fHbp and NHBA. The work indicates that mOMV from N. cinerea can be used as a platform to induce immune responses against antigens involved in the protective immune response against meningococcal and gonococcal diseases.

Structure of the malaria vaccine candidate Pfs48/45 and its recognition by transmission blocking antibodies.

An effective malaria vaccine remains a global health priority and vaccine immunogens which prevent transmission of the parasite will have important roles in multi-component vaccines. One of the most promising candidates for inclusion in a transmission-blocking malaria vaccine is the gamete surface protein Pfs48/45, which is essential for development of the parasite in the mosquito midgut. Indeed, antibodies which bind Pfs48/45 can prevent transmission if ingested with the parasite as part of the mosquito bloodmeal. Here we present the structure of full-length Pfs48/45, showing its three domains to form a dynamic, planar, triangular arrangement. We reveal where transmission-blocking and non-blocking antibodies bind on Pfs48/45. Finally, we demonstrate that antibodies which bind across this molecule can be transmission-blocking. These studies will guide the development of future Pfs48/45-based vaccine immunogens.

Poor CD4+ T Cell Immunogenicity Limits Humoral Immunity to P. falciparum Transmission-Blocking Candidate Pfs25 in Humans.

Plasmodium falciparum transmission-blocking vaccines (TBVs) targeting the Pfs25 antigen have shown promise in mice but the same efficacy has never been achieved in humans. We have previously published pre-clinical data related to a TBV candidate Pfs25-IMX313 encoded in viral vectors which was very promising and hence progressed to human clinical trials. The results from the clinical trial of this vaccine were very modest. Here we unravel why, contrary to mice, this vaccine has failed to induce robust antibody (Ab) titres in humans to elicit transmission-blocking activity. We examined Pfs25-specific B cell and T follicular helper (Tfh) cell responses in mice and humans after vaccination with Pfs25-IMX313 encoded by replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA) delivered in the heterologous prime-boost regimen via intramuscular route. We found that after vaccination, the Pfs25-IMX313 was immunologically suboptimal in humans compared to mice in terms of serum Ab production and antigen-specific B, CD4+ and Tfh cell responses. We identified that the key determinant for the poor anti-Pfs25 Ab formation in humans was the lack of CD4+ T cell recognition of Pfs25-IMX313 derived peptide epitopes. This is supported by correlations established between the ratio of proliferated antigen-specific CD4+/Tfh-like T cells, CXCL13 sera levels, and the corresponding numbers of circulating Pfs25-specific memory B cells, that consequently reflected on antigen-specific IgG sera levels. These correlations can inform the design of next-generation Pfs25-based vaccines for robust and durable blocking of malaria transmission.

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, 2 healthy malaria-naive United Kingdom adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI. Parasitemia developed in both volunteers, and prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high-quality genome assembly by using a hybrid assembly method. We analyzed leading vaccine candidate antigens and multigene families, including the vivax interspersed repeat (VIR) genes, of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.

Safety and Immunogenicity of ChAd63/MVA Pfs25-IMX313 in a Phase I First-in-Human Trial.

Background: Transmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector. Methods: Clinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points. Results: The reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay. Conclusion: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation. Trial Registration: Clinicaltrials.gov NCT02532049.

A Universal Plug-and-Display Vaccine Carrier Based on HBsAg VLP to Maximize Effective Antibody Response.

Development of effective malaria vaccines requires delivery platforms to enhance the immunogenicity and efficacy of the target antigens. This is particularly challenging for transmission-blocking malaria vaccines (TBVs), and specifically for those based on the Pfs25 antigen, that need to elicit very high antibody titers to stop the parasite development in the mosquito host and its transmission. Presenting antigens to the immune system on virus-like particles (VLPs) is an efficient way to improve the quantity and quality of the immune response generated. Here we introduce for the first time a new VLP vaccine platform, based on the well-established hepatitis B surface antigen (HBsAg) fused to the SpyCatcher protein, so that the antigen of interest, linked to the SpyTag peptide, can be easily displayed on it (Plug-and-Display technology). As little as 10% of the SpyCatcher::HBsAg VLPs decorated with Pfs25::SpyTag (molar ratio) induces a higher antibody response and transmission-reducing activity in mice compared to the soluble protein, with 50 and 90% of the VLP coupled to the antigen further enhancing the response. Importantly, using this carrier that is a vaccine antigen itself could be beneficial, as we show that anti-HBsAg IgG antibodies are induced without interfering with the Pfs25-specific immune response generated. Furthermore, pre-existing anti-HBsAg immunity does not affect the antigen-specific response to Pfs25::SpyTag-SpyCatcher::HBsAg, suggesting that these VLPs can have a broad use as a vaccine platform.

Combination of RTS,S and Pfs25-IMX313 Induces a Functional Antibody Response Against Malaria Infection and Transmission in Mice.

The last two decades saw a dramatic reduction in malaria incidence rates, but this decrease has been stalling recently, indicating control measures are starting to fail. An effective vaccine, particularly one with a marked effect on disease transmission, would undoubtedly be an invaluable tool for efforts to control and eliminate malaria. RTS,S/AS01, the most advanced malaria vaccine to date, targets the parasite before it invades the liver and has the potential to prevent malaria disease as well as transmission by preventing blood stage infection and therefore gametocytogenesis. Unfortunately efficacy in a phase III clinical trial was limited and it is widely believed that a malaria vaccine needed to contain multiple antigens from different life-cycle stages to have a realistic chance of success. A recent study in mice has shown that partially efficacious interventions targeting the pre-erythrocytic and the sexual lifecycle stage synergise in eliminating malaria from a population over multiple generations. Hence, the combination of RTS,S/AS01 with a transmission blocking vaccine (TBV) is highly appealing as a pragmatic and powerful way to increase vaccine efficacy. Here we demonstrate that combining Pfs25-IMX313, one of the TBV candidates currently in clinical development, with RTS,S/AS01 readily induces a functional immune response against both antigens in outbred CD1 mice. Formulation of Pfs25-IMX313 in AS01 significantly increased antibody titres when compared to formulation in Alhydrogel, resulting in improved transmission reducing activity in standard membrane feeding assays (SMFA). Upon co-formulation of Pfs25-IMX313 with RTS,S/AS01, the immunogenicity of both vaccines was maintained, and functional assessment of the induced antibody response by SMFA and inhibition of sporozoite invasion assay (ISI) showed no reduction in biological activity against parasites of both lifecycle stages. Should this findings be translatable to human vaccination this could greatly aid efforts to eliminate and eventually eradicate malaria.

Structural basis for recognition of the malaria vaccine candidate Pfs48/45 by a transmission blocking antibody.

The quest to develop an effective malaria vaccine remains a major priority in the fight against global infectious disease. An approach with great potential is a transmission-blocking vaccine which induces antibodies that prevent establishment of a productive infection in mosquitos that feed on infected humans, thereby stopping the transmission cycle. One of the most promising targets for such a vaccine is the gamete surface protein, Pfs48/45. Here we establish a system for production of full-length Pfs48/45 and use this to raise a panel of monoclonal antibodies. We map the binding regions of these antibodies on Pfs48/45 and correlate the location of their epitopes with their transmission-blocking activity. Finally, we present the structure of the C-terminal domain of Pfs48/45 bound to the most potent transmission-blocking antibody, and provide key molecular information for future structure-guided immunogen design.

IgG Responses to Porins and Lipopolysaccharide within an Outer Membrane-Based Vaccine against Nontyphoidal Salmonella Develop at Discordant Rates.

Antibodies acquired after vaccination or natural infection with Gram-negative bacteria, such as invasive Salmonella enterica serovar Typhimurium, can protect against disease. Immunization with naturally shed outer membrane vesicles from Gram-negative bacteria is being studied for its potential to protect against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification, and the resulting particles, generalized modules for membrane antigens (GMMA), not only offer potential as vaccines but also can facilitate the study of B-cell responses to bacterial antigens. Here we show that the response to immunization with GMMA from S Typhimurium (STmGMMA) provides B-cell-dependent protection and induces antibodies to two immunodominant antigens, lipopolysaccharide (LPS) and porins. Antibodies to LPS O antigen (O-Ag) markedly enhance protection in the spleen, but this effect is less marked in the liver. Strikingly, IgG responses to LPS and porins develop with distinct kinetics. In the first week after immunization, there is a dramatic T-cell-independent B1b-cell-associated induction of all IgG isotypes, except IgG1, to porins but not to LPS. In contrast, production of IgG1 to either antigen was delayed and T cell dependent. Nevertheless, after 1 month, cells in the bone marrow secreting IgG against porins or LPS were present at a similar frequency. Unexpectedly, immunization with O-Ag-deficient STmGMMA did not substantially enhance the anti-porin response. Therefore, IgG switching to all antigens does not develop synchronously within the same complex and so the rate of IgG switching to a single component does not necessarily reflect its frequency within the antigenic complex.IMPORTANCE Vaccines save millions of lives, yet for some infections there are none. This includes some types of Salmonella infections, killing hundreds of thousands of people annually. We show how a new type of vaccine, called GMMA, that is made from blebs shed from the Salmonella cell wall, works to protect against infection in mice by inducing host proteins (antibodies) specifically recognizing bacterial components (antigens). The rate of development of IgG antibody to antigens within GMMA occurred with different kinetics. However, the antibody response to GMMA persists and is likely to provide prolonged protection for those who need it. These results help show how antibody responses to bacterial antigens develop and how vaccines like GMMA can work and help prevent infection.

Contribution of factor H-Binding protein sequence to the cross-reactivity of meningococcal native outer membrane vesicle vaccines with over-expressed fHbp variant group 1.

Factor H-binding protein (fHbp) is an important meningococcal vaccine antigen. Native outer membrane vesicles with over-expressed fHbp (NOMV OE fHbp) have been shown to induce antibodies with broader functional activity than recombinant fHbp (rfHbp). Improved understanding of this broad coverage would facilitate rational vaccine design. We performed a pair-wise analysis of 48 surface-exposed amino acids involved in interacting with factor H, among 383 fHbp variant group 1 sequences. We generated isogenic NOMV-producing meningococcal strains from an African serogroup W isolate, each over-expressing one of four fHbp variant group 1 sequences (ID 1, 5, 9, or 74), including those most common among invasive African meningococcal isolates. Mice were immunised with each NOMV, and sera tested for IgG levels against each of the rfHbp ID and for ability to kill a panel of heterologous meningococcal isolates. At the fH-binding site, ID pairs differed by a maximum of 13 (27%) amino acids. ID 9 shared an amino acid sequence common to 83 ID types. The selected ID types differed by up to 6 amino acids, in the fH-binding site. All NOMV and rfHbp induced high IgG levels against each rfHbp. Serum killing from mice immunised with rfHbp was generally less efficient and more restricted compared to NOMV, which induced antibodies that killed most meningococci tested, with decreased stringency for ID type differences. Breadth of killing was mostly due to anti-fHbp antibodies, with some restriction according to ID type sequence differences. Nevertheless, under our experimental conditions, no relationship between antibody cross-reactivity and variation fH-binding site sequence was identified. NOMV over-expressing different fHbp IDs belonging to variant group 1 induce antibodies with fine specificities against fHbp, and ability to kill broadly meningococci expressing heterologous fHbp IDs. The work reinforces that meningococcal NOMV with OE fHbp is a promising vaccine strategy, and provides a basis for rational selection of antigen sequence types for over-expression on NOMV.