Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10.4-HspX

To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection including the persister bacteria, antigens of Mtb10.4 (Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) without affinity tag with potential advantage for clinical use. The human T-cell responses to MH were evaluated for its immunogenicity. Furthermore, MH was emulsified in an adjuvant composed of N,N′-dimethyl-N,N′-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalose-6,6-dimycolate (TDM) to construct subunit vaccine, whose immunogenicity and potency to boost BCG primed immunity against M. tuberculosis infection were evaluated in mice. The results showed that the fusion protein MH without affinity tag was stably produced in Escherichia coli and was successfully purified by chromatography. MH was strongly recognized by human T cells from TB patients and persons latently infected with M. tuberculosis. In conclusion, MH in adjuvant DDA-TDM generated strong antigen-specific humoral and cell-mediated immunity, and had the capability to enhance BCG-primed immunity and the protective efficacy against M. tuberculosis in mice. These findings suggest that MH in DDA-TDM have the potential to be a good multistage tuberculosis vaccine candidate.     

Development, expression, and murine testing of a multistage Plasmodium falciparum malaria vaccine candidate

A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.     

Development of a bivalent conjugate vaccine candidate against malaria transmission and typhoid fever

Immune responses to poorly immunogenic antigens, such as polysaccharides, can be enhanced by conjugation to carriers. Our previous studies indicate that conjugation to Vi polysaccharide of Salmonella Typhi may also enhance immunogenicity of some protein carriers. We therefore explored the possibility of generating a bivalent vaccine against Plasmodium falciparum malaria and typhoid fever, which are co-endemic in many parts of the world, by conjugating Vi polysaccharide, an approved antigen in typhoid vaccine, to Pfs25, a malaria transmission blocking vaccine antigen in clinical trials. Vi-Pfs25 conjugates induced strong immune responses against both Vi and Pfs25 in mice, whereas the unconjugated antigens are poorly immunogenic. Functional assays of immune sera revealed potent transmission blocking activity mediated by anti-Pfs25 antibody and serum bactericidal activity due to anti-Vi antibody. Pfs25 conjugation to Vi modified the IgG isotype distribution of antisera, inducing a Th2 polarized immune response against Vi antigen. This conjugate may be further developed as a bivalent vaccine to concurrently target malaria and typhoid fever.     

Progress toward a malaria vaccine

In initial human trials, synthetic vaccines have induced humoral immunity sufficient to prevent clinical infection in some cases and delay it in others. Progress in induction of cellular immunity is also noteworthy with identification of determinants recognized by T cells. Antigenic variation and consequent blunting of immunogenicity may not be as troublesome as feared.     

A novel design of a multi-antigenic,multistage and multi-epitope vaccine against Helicobacter pylori: An in silico approach

Helicobacter pylori have colonized the gastric mucosa of half of the population worldwide. This bacterium is classified as a definitive type I carcinogen by the World Health Organization and no effective vaccine has been found against it yet. Thus, a logical and rational vaccine design against H. pylori is necessary. Because of its tremendous complexity and elicited immune responses, the vaccine design should considered multiple antigens to enhance immune-protection, involved in the different stages of pathogenesis besides inducing a specific immune response by B- and T-cell multi-epitopes. In this study, emphasis was placed on the design of a new unique vaccine named CTB-multiHp. In silico techniques were used to design a chimeric construct consisting of cholera toxin B subunit fused to multi-epitope of urease B (residue 148–158, 188–198), cytotoxin-associated gene A (residue 584–602), neutrophil activating protein (residue 4–28), vacuolating cytotoxin gene A (residue 63–81), H. pylori adhesine A (residue77–99), heat shock protein A (residue 32–54) and gamma glutamyl transpeptidase (residue 271–293). The tertiary structure and features of the vaccine were analyzed. The chimeric protein was expressed in Escherichia coli BL21 and the serology analyses indicated that the CTB-multiHp protein produced exhibit immune-reactivity. The results showed that CTB-multiHp could be a good vaccine candidate against H. pylori. Ongoing studies will evaluate the effects of CTB-multiHp against H. pylori infection.     

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-γ-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-γ-producing CD8⁺ T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.     

Construction and evaluation of a live vaccine against Edwardsiella tarda and Vibrio harveyi: laboratory vs. mock field trial

Edwardsiella tarda and Vibrio harveyi are Gram-negative bacterial pathogens that affect a wide range of cultured fish. In previous studies, we have reported an E. tarda live vaccine ATCC15947 and a V. harveyi subunit vaccine DegQ. On the basis of these studies, in the present study we developed a cross protective vaccine against both E. tarda and V. harveyi by constructing a recombinant ATCC15947, Et15VhD, that expresses and secrets V. harveyi DegQ as a soluble antigen. Laboratory studies in a turbot (Scophthalmus maximus) model showed that Et15VhD elicited significant protections against E. tarda and V. harveyi when administered via intraperitoneal injection, oral feeding, immersion, and oral plus immersion, respectively. Microbiological analysis indicated dissemination and transient colonization of Et15VhD in fish tissues following vaccination. Since, compared to injection, oral plus immersion is a practically more acceptable vaccination procedure in aquaculture, we conducted a mock field trial to further examine the potential of Et15VhD as an oral plus immersion vaccine. The results showed that during the period before artificial bacterial challenge, mortality was observed in both the vaccinated group and the control group; however, the mortality of Et15VhD-vaccinated fish was significantly lower than that of the control fish. Following experimental challenge with E. tarda and V. harveyi at one and two months post-vaccination, Et15VhD-vaccinated fish exhibited dramatically increased survival rates compared to control fish. Serum antibody analysis indicated specific antibody production in Et15VhD-vaccinated fish. Taken together, these results demonstrate that Et15VhD induces strong protective immunity against E. tarda and V. harveyi under both laboratory and mock field conditions, which suggests a potential for Et15VhD to be used in aquaculture.     

HIV-1 AE重组型疫苗构建及免疫原性研究

目的利用AE亚型的膜蛋白,研发抗HIV-1AE重组型的疫苗并进行免疫原性评价。方法构建表达中国流行株97CNGX2F（HIV-1 AE2F）包膜蛋白gp140的重组痘苗病毒rVVT140AE。使用表达gp140的DNA疫苗和rVVT140AE用初免-加强方式免疫小鼠。免疫结束后第2周处死小鼠,分别检测特异性抗体滴度和特异性T细胞反应。结果本疫苗所活化的特异性抗体滴度在3 200和51 200之间,几何平均值为11 143;总T细胞免疫反应强度为（1 918±442）SFCs/10^6脾细胞,其中针对env1、env2、env3、env4肽池的免疫反应强度分别为（1 280±330）SFCs/10^6脾细胞,（66±16）SFCs/10^6脾细胞,（163±34）SFCs/10^6脾细胞和（409±96）SFCs/10^6脾细胞,均显著高于空载体对照组（〈10 SFCs/10^6脾细胞）。结论本实验构建的HIV-1 AE2F株包膜蛋白gp140重组痘苗病毒疫苗可作为针对我国HIV-1流行株的候选疫苗免疫原。     

Immunoinformatics approach for multi-epitope vaccine design against P. falciparum malaria

Plasmodium falciparum (P. falciparum) is a leading causative agent of malaria, an infectious disease that can be fatal. Unfortunately, control measures are becoming less effective over time. A vaccine is needed to effectively control malaria and lead towards the total elimination of the disease. There have been multiple attempts to develop a vaccine, but to date, none have been certified as appropriate for wide-scale use. In this study, an immunoinformatics method is presented to design a multi-epitope vaccine construct predicted to be effective against P. falciparum malaria. This was done through the prediction of 12 CD4+ T-cell, 10 CD8+ T-cell epitopes and, 1 B-cell epitope which were assessed for predicted high antigenicity, immunogenicity, and non-allergenicity through in silico methods. The Human Leukocyte Antigen (HLA) population coverage showed that the alleles associated with the epitopes accounted for 78.48% of the global population. The CD4+ and CD8+ T-cell epitopes were docked to HLA-DRB1*07:01 and HLA-A*32:01 successfully. Therefore, the epitopes were deemed to be suitable as components of a multi-epitope vaccine construct. Adjuvant RS09 was added to the construct to generate a stronger immune response, as confirmed by an immune system simulation. Finally, the structural stability of the predicted multi-epitope vaccine was assessed using molecular dynamics simulations. The results show a promising vaccine design that should be further synthesised and assessed for its efficacy in an experimental laboratory setting.     

Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice.

Multiple antigen peptides (MAPs) containing epitopes of the major surface protein of the malaria sporozoite, the circumsporozoite (CS) protein, have been shown in previous studies to elicit antibody-mediated protection against sporozoite challenge in experimental murine and simian hosts. For the preparation for a phase I trial of a P. falciparum (T1B)4 MAP, which contains T and B cell epitopes from the CS repeat region, pre-clinical immunogenicity and adjuvant formulation studies were carried out in mice and Aotus monkeys. The (T1B)4 MAP was found to be immunogenic in three different species of owl monkeys, Aotus nancymae, A. vociferans and A. nigriceps. Optimal antibody responses were obtained in A. nancymae immunized s.c. with (T1B)4 MAP emulsified in Freund's, in which peak titers of over 10(6) were obtained in individual monkeys. MAP immunized A. vociferans also developed high levels of anti-sporozoite antibodies, although the kinetics and the magnitude of the response differed from A. nancymae. (T1B)4 MAP adsorbed to alum (aluminum hydroxide), a formulation that is acceptable for human use, was less immunogenic in naive A. nancymae, as well as A. nigriceps. The injection of MAPs/alum, however, significantly enhanced antibody responses in sporozoite-primed monkeys, suggesting that the administration of the MAP vaccine may be an effective means to increase the low levels of antibody present in individuals living in malaria endemic areas. The addition of a co-adjuvant QS-21, a purified saponin, significantly increased the immunogenicity of the alum-adsorbed MAP in both mice and monkeys, providing a vaccine formulation suitable for phase I trials in human volunteers.     

Comparative efficacy of pre-erythrocytic whole organism vaccine strategies against the malaria parasite

Despite major efforts over the past 50 years to develop a malaria vaccine, no product has been licensed yet. Irradiated sporozoites are the benchmark for an experimental live-attenuated malaria vaccine that induces potent protection against re-infection in humans and animal models. Lasting protection can also be elicited by parasite attenuation via tailored genetic modification or drug cover leading to renewed interest in whole-organism vaccination strategies. In this study, we systematically compared the protective efficacy of different whole-organism vaccination approaches in the Plasmodium berghei/C57bl/6 rodent malaria model. We applied blood stage parasitemia and quantitative RT-PCR of liver parasite loads as two complementary primary endpoints of a malaria challenge infection. We were able to demonstrate similar potency of genetic attenuation, i.e., uis3(−) and p36p(−) parasites, and prophylactic drug cover, i.e., azithromycin, pyrimethamine, primaquine and chloroquine, during sporozoite exposure in comparison to irradiated sporozoites. Importantly, when animals were covered with the antibiotic azithromycin during sporozoite exposure we observed superior protection. On the other end, immunizations with heat-killed and over-irradiated sporozoites failed to confer any detectable protection. Together, we show that systematic pre-clinical evaluation and quantification of vaccine efficacy is vital for identification of the most potent whole organism anti-malaria vaccine strategy.     

The main obstacle to a malaria vaccine: the malaria parasite

The challenge to development of a malaria vaccine will be the variability of the sequences of target antigens. We discuss the variability in the T cell epitopes on the circumsporozoite protein of Plasmodium falciparum and the rapid selection of mutants after immunization with a protein from the asexual erythrocytic stage of P. knowlesi.     

紧急接种甲乙肝联合疫苗和乙脑疫苗的安全性评价

目的评价甲乙肝联合疫苗和乙型脑膜炎减毒活疫苗安全性以及免疫效果。方法把甲乙肝联合疫苗同乙型脑膜炎减毒活疫苗同时接种于洁净级小白鼠后观察不良反应,并在45 d后对其做免疫原性的检查和病理组织学观察。结果接种甲乙肝联合疫苗和乙型脑膜炎减毒活疫苗组小白鼠均无毒副作用产生,且都产生免疫,肝脏和小脑的病理切片均无病变产生。结论同时接种甲乙肝联合疫苗和乙型脑膜炎减毒活疫苗安全、有效。     

Protection against malaria by immunization with a Plasmodium yoelii circumsporozoite protein nucleic acid vaccine

Nucleic acid vaccines provide an exciting new alternative approach to developing the multiantigen vaccines designed to induce protective antibody and T-cell responses against Plasmodium proteins that many experts believe will be required for effective protection against malaria. As a first step in this process, we produced a plasmid DNA vaccine that includes the gene encoding the P. Yoelii circumsporozoite protein (PyCSP). This vaccine induced higher levels of antibodies and cytotoxic T lymphocytes against PyCSP than immunization with irradiated sporozoites, and protected 9 of the first 16 mice immunized. Work is now in progress to optimize immunization regimens, establish the mechanisms of protective immunity induced by the vaccine, and to determine whether protective immunity can be increased by vaccinating with multiple nucleic acid vaccines designed to produce immune responses against multiple targets.     

Construction and evaluation of a chimeric pseudoinfectious virus vaccine to prevent Japanese encephalitis

Multiple vaccines exist to control Japanese encephalitis (JE), but all suffer from problems. We have developed a new type of flavivirus vaccine, a pseudoinfectious virus (RepliVAX WN) that prevents West Nile virus (WNV)-induced disease. Here, we describe production of a chimeric RepliVAX (RepliVAX JE) that expresses the JE virus (JEV) prM and E proteins. Our prototype RepliVAX JE replicated poorly in cells, but blind passage produced a better-growing derivative, and analyses of this derivative allowed us to engineer a second-generation RepliVAX (RepliVAX JE.2) that grew to high titers. RepliVAX JE.2 elicited neutralizing antibodies in both mice and hamsters and provided 100% protection from a lethal challenge with JEV or WNV, respectively. These results demonstrate the utility our RepliVAX platform for producing a JE vaccine.     

Immunological and physical evaluation of the multistage tuberculosis subunit vaccine candidate H56/CAF01 formulated as a spray-dried powder

Liquid vaccine dosage forms have limited stability and require refrigeration during their manufacture, distribution and storage. In contrast, solid vaccine dosage forms, produced by for example spray drying, offer improved storage stability and reduced dependence on cold-chain facilities. This is advantageous for mass immunization campaigns for global public health threats, e.g., tuberculosis (TB), and offers cheaper vaccine distribution. The multistage subunit vaccine antigen H56, which is a fusion protein of the Mycobacterium tuberculosis (Mtb) antigens Ag85B, ESAT-6, and Rv2660, has been shown to confer protective efficacy against active TB before and after Mtb exposure in preclinical models, and it is currently undergoing clinical phase 2a testing. In several studies, including a recent study comparing multiple clinically relevant vaccine adjuvants, the T helper type 1 (Th1)/Th17-inducing adjuvant CAF01 was the most efficacious adjuvant for H56 to stimulate protective immunity against Mtb. With the long-term goal of designing a thermostable and self-administrable dry powder vaccine based on H56 and CAF01 for inhalation, we compared H56 spray-dried with CAF01 with the non-spray-dried H56/CAF01 vaccine with respect to their ability to induce systemic Th1, Th17 and humoral responses after subcutaneous immunization. Here we show that spray drying of the H56/CAF01 vaccine results in preserved antigenic epitope recognition and adjuvant activity of CAF01, and the spray-dried, reconstituted vaccine induces antigen-specific Th1, Th17 and humoral immune responses, which are comparable to those stimulated by the non-spray-dried H56/CAF01 vaccine. In addition, the spray-dried and reconstituted H56/CAF01 vaccine promotes similar polyfunctional CD4⁺ T-cell responses as the non-spray-dried vaccine. Thus, our study provides proof-of-concept that spray drying of the subunit vaccine H56/CAF01 preserves vaccine-induced humoral and cell-mediated immune responses. These results support our ongoing efforts to develop a thermostable, dry powder-based TB vaccine.     

Construction and immunogenicity of a T cell epitope-based subunit vaccine candidate against Mycobacterium tuberculosis

Despite antibiotic treatment and Bacille Calmette-Guérin (BCG) vaccination, Mycobacterium tuberculosis remains a major public health burden in most developing countries. Therefore, developing an improved vaccine is high priority. In this study, we cloned the genes of the immunodominant antigen of M. tuberculosis viz. its 38-kDa antigen (Pst homolog) (Rv0934, PstS1), and its T cell epitopes (amino acid [aa]169–405 and [aa]802–1119), which we termed PstS1p. Prokaryotic expression showed that the two recombinant proteins were mainly in the form of inclusion bodies. We also evaluated the immunity and immunogenicity of PstS1 and PstS1p. Both PstS1 and its T cell epitopes elicited significantly higher antigen-specific immunoglobulin G (IgG) antibodies in mouse serum, indicating that they enhanced antibody response. They also elicited the T helper 1 (Th1)-type response and promoted CD4⁺ T cell proliferation. Compared to PstS1, PstS1p promoted stronger cell-mediated immune response. These data indicate that PstS1p is highly immunogenic in mice, and may be a promising candidate vaccine for controlling tuberculosis.