The challenges and opportunities for the development of a T-cell epitope-based herpes simplex vaccine

Herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) infections have been prevalent since the ancient Greek times. To this day, they still affect a staggering number of over a billion individuals worldwide. HSV-1 infections are predominant than HSV-2 infections and cause potentially blinding ocular herpes, oro-facial herpes and encephalitis. HSV-2 infections cause painful genital herpes, encephalitis, and death in newborns. While prophylactic and therapeutic HSV vaccines remain urgently needed for centuries, their development has been difficult. During the most recent National Institute of Health (NIH) workshop titled “Next Generation Herpes Simplex Virus Vaccines: The Challenges and Opportunities”, basic researchers, funding agencies, and pharmaceutical representatives gathered: (i) to assess the status of herpes vaccine research; and (ii) to identify the gaps and propose alternative approaches in developing a safe and efficient herpes vaccine. One “common denominator” among previously failed clinical herpes vaccine trials is that they either used a whole virus or a whole viral protein, which contain both “pathogenic symptomatic” and “protective asymptomatic” antigens and epitopes. In this report, we continue to advocate developing “asymptomatic” epitope-based sub-unit vaccine strategies that selectively incorporate “protective asymptomatic” epitopes which: (i) are exclusively recognized by effector memory CD4⁺ and CD8⁺ T cells (T_EM cells) from “naturally” protected seropositive asymptomatic individuals; and (ii) protect human leukocyte antigen (HLA) transgenic animal models of ocular and genital herpes. We review the role of animal models in herpes vaccine development and discuss their current status, challenges, and prospects.     

Characterization of a multi-lipopeptides mixture used as an HIV-1 vaccine candidate.

A multi-component vaccine has been defined, which contains six different synthetic 24- to 32-amino acid lipopeptides derived from the sequence of HIV-1 proteins. The physicochemical properties of the lipopeptide components were compatible with multi-dimensional analysis, using RP-HPLC, Edman sequencing, electrospray mass spectrometry, and 2D-NMR. Detailed analysis of the impurity profiles led to the detection and evaluation of the relative proportions of most by-products: several contaminants resulted from the formation of acetylated fragments, transpeptidation reactions with succinimide or piperidide formation, or methionine and/or tryptophan mono-oxidations. The first batch to be produced underwent extensive pharmacotoxicological testings to confirm its safety; this vaccine candidate has now been used in phase I clinical trials. Despite the complexity of such multi-lipopeptide vaccines, our findings suggest the possibility of preparing a clear and precise assignment of by-products to toxicologically qualified impurities in the eventuality of a future production of several successive batches.     

Prospects for a virus non-structural protein as a subunit vaccine

The design of contemporary vaccines to date has been dependent upon our understanding of the process of stimulation of protective immunity by virion proteins. However, studies with the flaviviruses have demonstrated that protective immunity can be elicited by a non-structural protein, NS1. Surprisingly, immunity to infection is stimulated in the absence of neutralizing antibodies. The information concerning NS1-induced immunity is described and is discussed with relevance to the role of the protein in immunity and vaccine development, against flavivirus diseases. The possibility exists that the phenomenon of non-structural protein induced immunity is applicable to the prophylaxis of other virus diseases.     

Policy making for vaccine use as a driver of vaccine innovation and development in the developed world

In the past 200 years, vaccines have had unmistakable impacts on public health including declines in morbidity and mortality, most markedly in economically-developed countries. Highly engineered vaccines including vaccines for conditions other than infectious diseases are expected to dominate future vaccine development. We examine immunization vaccine policy as a driver of vaccine innovation and development. The pathways to recommendation for use of licensed vaccines in the US, UK, Canada and Australia have been similar, including: expert review of disease epidemiology, disease burden and severity; vaccine immunogenicity, efficacy and safety; programmatic feasibility; public demand; and increasingly cost-effectiveness. Other attributes particularly important in development of future vaccines are likely to include: duration of immunity for improved vaccines such as pertussis; a greater emphasis on optimizing community protection rather than direct protection only; programmatic implementation, feasibility, improvements (as in the case of development of a universal influenza vaccine); public concerns/confidence/fears related to outbreak pathogens like Ebola and Zika virus; and major societal burden for combating hard to treat diseases like HIV and antimicrobial resistant pathogens. Driving innovation and production of future vaccines faces enormous economic hurdles as available approaches, technologies and regulatory pathways become more complex. As such, cost-mitigating strategies and focused, aligned efforts (by governments, private organizations, and private-public partnerships) will likely be needed to continue to spur major advances in vaccine technologies and development.     

Nutrient deficit as a possible prerequisite in the development of poisoning by small doses of lead

A reduced consumption of essential elements (like vegetable fat, calcium, and vitamin C) by children, aged 5, could aggravate the effect exerted by lead on the structure and functions of erythrocyte membranes. In the end, it could be a precondition increasing the risk of intoxication by the impact of small doses of lead coming from the environment.     

Strategies for the development of an antimalarial vaccine.

Susceptible Aotus monkeys were immunized with Escherichia coli-derived fusion proteins containing partial sequences of the proteins MSAI, SERP, HRPII and with a group of three recombinant antigens isolated by screening with an antiserum raised against the protective 41 kDa protein band. HRPII, the combination of the fusion proteins of the 41 kDa group and a mixture of two sequences of SERP conferred significant protection against a challenge infection with Plasmodium falciparum blood stages. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences. In two independent immunization trials it was shown that immunization of Aotus monkeys with either of the two hybrid proteins can protect the animals from an experimental P. falciparum infection.     

Epitope mapping immunodominant regions of the PilA protein of nontypeable Haemophilus influenzae (NTHI) to facilitate the design of two novel chimeric vaccine candidates

We designed and tested three PilA-derived vaccine candidates in a chinchilla model of ascending nontypeable Haemophilus influenzae (NTHI)-induced otitis media (OM). Delivery of antiserum directed against each immunogen conferred varying degrees of protection. Presentation of a B-cell epitope derived from the OMP P5 adhesin at the N-terminus of recombinant soluble PilA protein (as opposed to the C-terminus), resulted in a protective chimeric immunogen that combined epitopes from two distinct NTHI adhesins (type IV pili and OMP P5). Incorporating protective epitopes derived from two NTHI adhesins/virulence determinants into a single pediatric vaccine candidate to prevent OM has multiple potential inherent advantages.     

Fused Mycobacterium tuberculosis multi-stage immunogens with an Fc-delivery system as a promising approach for the development of a tuberculosis vaccine

Tuberculosis (TB) remains a major health problem worldwide. Currently, the Bacilli Calmette-Guérin (BCG) is the only available licensed TB vaccine, which has low efficacy in protection against adult pulmonary TB. Therefore, the development of a safe and effective vaccine against TB needs global attention. In the present study, a novel multi-stage subunit vaccine candidate from culture filtrate protein-10 (CFP-10) and heat shock protein X (HspX) of Mycobacterium tuberculosis fused to the Fc domain of mouse IgG2a as a selective delivery system for antigen-presenting cells (APCs) was produced and its immunogenicity assessed. The optimized gene constructs were introduced into pPICZαA expression vectors, and the resultant plasmids (pPICZαA-CFP-10:Hspx:Fcγ2a and pPICZαA-CFP-10:Hspx:His) were transferred into Pichia pastoris by electroporation. The identification of both purified recombinant fusion proteins was evaluated by SDS-PAGE and immunoblotting. Then the immunogenicity of the recombinant proteins with and without BCG was evaluated in BALB/c mice by assessing the level of IFN-γ, IL-12, IL-4, IL-17 and TGF-β cytokines. Both multi-stage vaccines (CFP-10:HspX:Fcγ2a and CFP-10:HspX:His) induced Th1-type cellular responses by producing high level of IFN-γ (272 pg/mL, p < 0.001) and IL-12 (191 pg/mL, p < 0.001). However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low level of IL-4 (10 pg/mL) compared to the CFP-10:HspX:His group. The production of IFN-γ to CFP-10:HspX:Fcγ2a was markedly consistent and showed an increasing trend for IL-12 compared with the BCG or CFP-10:HspX:His primed and boosted groups. Findings revealed that CFP-10:Hspx:Fcγ2a fusion protein can elicit strong Th1 antigen-specific immune responses in favor of protective immunity in mice and could provide new insight for introducing an effective multi-stage subunit vaccine against TB.     

Systemic immunization with an epitope-based vaccine elicits a Th1-biased response and provides protection against Helicobacter pylori in mice

Vaccine-mediated Th1-biased CD4+ T cell responses have been shown to be crucial for protection against Helicobacter pylori (H. pylori). In this study, we investigated whether a vaccine composed of CD4+ T cell epitopes together with Th1 adjuvants could confer protection against H. pylori in a mouse model. We constructed an epitope-based vaccine, designated Epivac, which was composed of predicted immunodominant CD4+ T cell epitopes from H. pylori adhesin A (HpaA), urease B (UreB) and cytotoxin-associated gene A product (CagA). Together with four different Th1 adjuvants, Epivac was administered subcutaneously and the prophylactic potential was examined. Compared to non-immunized mice, immunization with Epivac alone or with a Th1 adjuvant significantly reduced H. pylori colonization, and better protection was observed when an adjuvant was used. Immunized mice exhibited a strong local and systemic Th1-biased immune response, which may contribute to the inhibition of H. pylori colonization. Though a significant specific antibody response was induced by the vaccine, no correlation was found between the intensity of the humoral response and the protective effect. Our results suggest that a vaccine containing CD4+ T cell epitopes is a promising candidate for protection against H. pylori infection.     

The non-toxic and biodegradable adjuvant Montanide ISA 720/CpG can replace Freund's in a cancer vaccine targeting ED-B--a prerequisite for clinical development

We have recently shown that immunization against the extra domain-B (ED-B) of fibronectin, using Freund's adjuvant, reduces tumor growth in mice by 70%. In the present study we compare the immune response generated against ED-B using the non-toxic and biodegradable adjuvant Montanide ISA 720/CpG with the response elicited by Freund's adjuvant. Montanide ISA 720/CpG induced anti-ED-B antibodies with higher avidity and less variable levels between individuals than Freund's. Moreover, the duration of the immune response was longer and the generation of anti-ED-B antibodies in naïve mice was faster, when Montanide ISA 720/CpG was used. We conclude that it is possible to replace the mineral oil based adjuvant Freund's with an adjuvant acceptable for human use, which is a prerequisite for transfer of the ED-B vaccine to the clinic.     

Causal knowledge as a prerequisite for confounding evaluation: an application to birth defects epidemiology.

Common strategies to decide whether a variable is a confounder that should be adjusted for in the analysis rely mostly on statistical criteria. The authors present findings from the Slone Epidemiology Unit Birth Defects Study, 1992-1997, a case-control study on folic acid supplementation and risk of neural tube defects. When statistical strategies for confounding evaluation are used, the adjusted odds ratio is 0.80 (95% confidence interval: 0.62, 1.21). However, the consideration of a priori causal knowledge suggests that the crude odds ratio of 0.65 (95% confidence interval: 0.46, 0.94) should be used because the adjusted odds ratio is invalid. Causal diagrams are used to encode qualitative a priori subject matter knowledge.     

Characterization of the physical development of schoolchildren in different regions of Russia

Age-related changes in some physical development (PD) indices (height, weight, chest circumference, hand dynamometry, and vital capacity) were studied in 21,500 schoolchildren aged 7 to 17 years in 5 federal areas (Central, North-western, Urals, Southern, and Siberian) of the Russian Federation. The moderate development of both morphological and functional characteristics is prevalent in the school-children of all age groups, the proportion of their low development being higher than that of their high development. The proportion of children with average weight is virtually constant in age groups of 7 to 17 years and it is about 50% of the sample. The moderate level of other characteristics is noted for age-related variations of different intensity, which are more pronounced in girls. In terms of age, the functional parameters of PD are more variable than morphological ones. Age-related variation of the ratio of PD levels may be entirely assigned due to: a) heterochronicity of processes of increasing morphological parameters (longitudinal and latitudinal); b) a great variability of functional parameters. Recommendations on correction of schoolchildren's PD with physical educational means are given.     

HIV感染者B细胞的特点及其对疾病进展的影响

目的探讨浙江省台州市HIV扩大检测策略的影响因素,为完善当前HIV扩大检测策略提供依据。方法选取HIV扩大检测策略涉及的供需双方人员作为访谈对象,通过焦点组访谈和个体深入访谈的方式收集数据和信息,利用Nvivo 8.0软件分析影响因素。结果影响台州市需方进行HIV扩大检测的因素包括社会歧视、HIV检测内涵和有关政策知晓率低、HIV相关知识宣传滞后等;影响供方提供HIV扩大检测策略的因素包括资源投入不足、检测部门缺乏合作、隐私保护不充分、宣传不足等。结论应通过完善资源投入与配置机制,注重检测效率机制构建,动员社会参与,创新HIV检测技术等促进HIV扩大检测策略的实施,提高检测率。     

Alpha-C-galactosylceramide as an adjuvant for a live attenuated influenza virus vaccine

There is a substantial need to develop better influenza virus vaccines that can protect populations that are not adequately protected by the currently licensed vaccines. While live attenuated influenza virus vaccines induce superior immune responses compared to inactivated vaccines, the manufacturing process of both types of influenza virus vaccines is time consuming and may not be adequate during a pandemic. Adjuvants would be particularly useful if they could enhance the immune response to live attenuated influenza virus vaccines so that the amount of vaccine needed for a protective dose could be reduced. The glycolipid, alpha-galactosylceramide (alpha-GalCer), has recently been shown to have adjuvant activity for both inactivated and replicating recombinant vaccines. The goal of these experiments was to determine whether a derivative of alpha-GalCer, alpha-C-galactosylceramide (alpha-C-GalCer) can enhance the immune response elicited by a live attenuated influenza virus vaccine containing an NS1 protein truncation and reduce the amount of vaccine required to provide protection after challenge. Our results indicated that the adjuvant reduced both morbidity and mortality in BALB/c mice after challenge with wild type influenza virus. The adjuvant also increased the amount of influenza virus specific total IgG, IgG1, and IgG2a antibodies as well as IFN-γ secreting CD8⁺ T cells. By using knockout mice that are not able to generate NKT cells, we were able to demonstrate that the mechanism of adjuvant activity is dependent on NKT cells. Thus, our data indicate that stimulators of NKT cells represent a new avenue of adjuvants to pursue for live attenuated virus vaccines.     

Trypanosome microtubule-associated protein p15 as a vaccine for the prevention of African sleeping sickness

Trypanosomes cause African sleeping sickness, affecting millions of humans and animals. We tested trypanosome microtubule-associate protein (MAP p15) as a vaccine in mice, and show that p15 (native or recombinant) generated up to 100% protection from an otherwise lethal challenge of a heterologous strain of Trypanosoma brucei. We also tested the adenovirus as a vaccine delivery system and show that both adenoviral vector containing p15 gene or control adenovirus containing lacZ gene generated a protective response and exhibited strong CD8+ T-cell proliferation. These results suggest that the p15 protein itself is an effective vaccine and that the adenovirus may be used to mount a non-specific cellular immune response.     

Nucleic acid (DNA) immunization as a platform for dengue vaccine development

Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.     

Characterization of a Vero cell-adapted virulent strain of enterovirus 71 suitable for use as a vaccine candidate

Enterovirus 71 (EV71) is a neurotrophic virus that causes seasonal morbidity and mortality in children throughout the world with increasing frequency in recent years. Because of the lack of an effective antiviral agent, primary prevention, including the development of effective vaccines, is a top priority in terms of control strategies. Poliovirus vaccine technology, both live attenuated and inactivated, killed virus vaccines, can be adopted for use with EV71 because of their relatedness. In this study, we have characterized a laboratory-adapted EV71 strain, YN3-4a, which exhibits different characteristics from those of its parent isolate, neu, in having a rapid growth rate in Vero cells, a larger plaque size, and a lower LD(50) in newborn mice. The YN3-4a can be produced at a high viral titer of up to 10(10) tissue culture infective dose (TCID(50)) when grown in Vero cells, an approved substrate for virus vaccine production. Mouse antiserum raised against YN3-4a can neutralize a broad range of strains of EV71 isolated at different times from a variety of geographic regions. On passage in Vero cells, YN3-4a remained genetically and phenotypically stable. Many of the above-described features, such as high viral yield, strong immunogenicity, broad-based antigenic coverage, and passage stability, are desirable features in a prototype virus for the development of an inactivated viral vaccine.     

Current status and prospects for development of an HSV vaccine

Herpes simplex virus type 2 (HSV-2) infects 530 million people, is the leading cause of genital ulcer disease, and increases the risk of HIV-1 acquisition. Although several candidate vaccines have been promising in animal models, prophylactic and therapeutic vaccines have not been effective in clinical trials thus far. Null results from the most recent prophylactic glycoprotein D2 subunit vaccine trial suggest that we must reevaluate our approach to HSV-2 vaccine development. We discuss HSV-2 pathogenesis, immunity, and vaccine efforts to date, as well as the current pipeline of candidate vaccines and design of trials to evaluate new vaccine constructs.