Of mice and not humans: how reliable are animal models for evaluation of herpes CD8(+)-T cell-epitopes-based immunotherapeutic vaccine candidates?

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2)-specific CD8⁺ T cells that reside in sensory ganglia, appear to control recurrent herpetic disease by aborting or reducing spontaneous and sporadic reactivations of latent virus. A reliable animal model is the ultimate key factor to test the efficacy of therapeutic vaccines that boost the level and the quality of sensory ganglia-resident CD8⁺ T cells against spontaneous herpes reactivation from sensory neurons, yet its relevance has been often overlooked. Herpes vaccinologists are hesitant about using mouse as a model in pre-clinical development of therapeutic vaccines because they do not adequately mimic spontaneous viral shedding or recurrent symptomatic diseases, as occurs in human. Alternatives to mouse models are rabbits and guinea pigs in which reactivation arise spontaneously with clinical herpetic features relevant to human disease. However, while rabbits and guinea pigs develop spontaneous HSV reactivation and recurrent ocular and genital disease none of them can mount CD8⁺ T cell responses specific to Human Leukocyte Antigen- (HLA-)restricted epitopes. In this review, we discuss the advantages and limitations of these animal models and describe a novel “humanized” HLA transgenic rabbit, which shows spontaneous HSV-1 reactivation, recurrent ocular disease and mounts CD8⁺ T cell responses to HLA-restricted epitopes. Adequate investments are needed to develop reliable preclinical animal models, such as HLA class I and class II double transgenic rabbits and guinea pigs to balance the ethical and financial concerns associated with the rising number of unsuccessful clinical trials for therapeutic vaccine formulations tested in unreliable mouse models.     

A Vaxfectin-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes

Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin^®. Prophylactic immunization with Vaxfectin^®-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin^®-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.     

Nasal and skin delivery of IC31(®)-adjuvanted recombinant HSV-2 gD protein confers protection against genital herpes

Genital herpes caused by herpes simplex virus type 2 (HSV-2) remains the leading cause of genital ulcers worldwide. Given the disappointing results of the recent genital herpes vaccine trials in humans, development of novel vaccine strategies capable of eliciting protective mucosal and systemic immune responses to HSV-2 is urgently required. Here we tested the ability of the adjuvant IC31^® in combination with HSV-2 glycoprotein D (gD) used through intranasal (i.n.), intradermal (i.d.), or subcutaneous (s.c.) immunization routes for induction of protective immunity against genital herpes infection in C57BL/6 mice. Immunization with gD plus IC31^® through all three routes of immunization developed elevated gD-specific serum antibody responses with HSV-2 neutralizing activity. Whereas the skin routes promoted the induction of a mixed IgG2c/IgG1 isotype profile, the i.n. route only elicited IgG1 antibodies. All immunization routes were able to induce gD-specific IgG antibody responses in the vaginas of mice immunized with IC31^®-adjuvanted gD. Although specific lymphoproliferative responses were observed in splenocytes from mice of most groups vaccinated with IC31^®-adjuvanted gD, only i.d. immunization resulted in a significant splenic IFN-γ response. Further, immunization with gD plus IC31^® conferred 80–100% protection against an otherwise lethal vaginal HSV-2 challenge with amelioration of viral replication and disease severity in the vagina. These results warrant further exploration of IC31^® for induction of protective immunity against genital herpes and other sexually transmitted infections.     

Intranasal immunization with a proteoliposome-derived cochleate containing recombinant gD protein confers protective immunity against genital herpes in mice

The purpose of this study was to investigate the potential of intranasal (IN) immunization with Neisseria meningitides B proteoliposome (AFPL1) and AFPL1-derived cochleate (AFCo1), containing glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) for induction of protective immunity against genital herpes infection in mice. We could show that IN immunization with both AFPL1 and AFCo1 containing gD induced gD-specific IgG antibody and lymphoproliferative responses. However, IFN-γ response could only be detected in CD4⁺ splenic cells and genital lymph node cells of the AFCo1gD immunized mice upon recall antigen stimulation in vitro. Importantly, IN immunization with AFCo1gD could elicit a complete protection against an otherwise lethal vaginal challenge with HSV-2, while the AFPL1gD immunized mice were only partially protected. Further, we could show that the IFN-γ response and protective immunity observed after IN immunization with AFCo1gD are mediated via the adaptor molecule myeloid differentiation factor 88. These data may have implications for the development of a mucosal vaccine against genital herpes.     

An ocular mucosal administration of nanoparticles containing DNA vaccine pRSC-gD-IL-21 confers protection against mucosal challenge with herpes simplex virus type 1 in mice

Herpes stromal keratitis (HSK) is a chronic inflammatory process caused by the infection of herpes simplex virus type 1 (HSV-1). Development of a HSV-1 vaccine is a priority because these infections are common and cannot be well prevented. It appears that the potential of nanocarriers in DNA vaccination will be required to augment the immune response to DNA vaccines. Therefore, in the study, nanoparticles Fe₃O₄ coated with glutamic acid, DNA vaccine pRSC-gD-IL-21 and polyethylenimine were prepared and immunized in the mice by ocular mucosal administration. The immune responses and protection efficiency against HSV-1 challenge were also tested. The results showed that the nanoparticles containing DNA vaccine pRSC-gD-IL-21 induced mice to generate higher levels of specific neutralizing antibody, sIgA in tears, and IFN-γ, IL-4 in serum, and to enhance the cytotoxicities of NK cells and splenocytes as well as splenocyte proliferative response to glycoprotein D compared with those of the control mice. More importantly, the mice immunized with the experimental vaccine showed less HSK degree than that of the control mice after HSV-1 challenge of the murine ocular mucosa. In conclusion, an ocular mucosal administration of nanoparticles containing DNA vaccine confers strong specific immune responses and effective inhibition of HSK in a HSV-1 infected murine model.     

Lack of neonatal Fc receptor does not diminish the efficacy of the HSV-1 0ΔNLS vaccine against ocular HSV-1 challenge

The neonatal Fc receptor (FcRn) is constitutively expressed in the cornea and is up-regulated in response to herpes simplex virus type 1 (HSV-1). Previously, we found targeting cornea FcRn expression by small interfering RNA-mediated knockdown reduced the local efficacy of HSV-1 0ΔNLS vaccinated C57BL/6 mice against ocular challenge with HSV-1. The current study was undertaken to evaluate the HSV-1 0ΔNLS vaccine efficacy in FcRn deficient (FcRn KO) mice challenged with HSV-1. Whereas there was little neutralizing antibody detected in the serum of HSV-1 0ΔNLS vaccinated FcRn KO mice, these mice exhibited the same degree of protection against ocular challenge with HSV-1 as wild type (WT) C57BL/6 mice as measured by cumulative survival, infectious virus shed or retained in tissue, and corneal pathology including opacity and neovascularization. Mock-vaccinated FcRn KO mice were found to be more sensitive to ocular HSV-1 infection compared to mock-vaccinated (WT) mice in terms of cumulative survival and virus shedding. In addition, the FcRn KO mice generated significantly fewer effector (CD3⁺CD44⁺CD62L^-) and central (CD3⁺CD44⁺CD62L⁺) memory CD8⁺ T cells compared to the WT mice 7 days post infection. Collectively, mock-vaccinated FcRn KO mice are susceptible to ocular HSV-1 infection but HSV-1 0ΔNLS vaccinated FcRn KO mice are resistant suggesting that in addition to the FcRn, other pathways are involved in mediating the protective effect of the HSV-1 0ΔNLS vaccine against subsequent HSV-1 challenge.     

The adjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome–DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2 + CLDC was compared to gD2 + MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.     

Biologic interactions between HSV-2 and HIV-1 and possible implications for HSV vaccine development

Development of a safe and effective vaccine against herpes simplex virus type 2 (HSV-2) has the potential to limit the global burden of HSV-2 infection and disease, including genital ulcer disease and neonatal herpes, and is a global sexual and reproductive health priority. Another important potential benefit of an HSV-2 vaccine would be to decrease HIV infections, as HSV-2 increases the risk of HIV-1 acquisition several-fold. Acute and chronic HSV-2 infection creates ulcerations and draws dendritic cells and activated CD4+ T cells into genital mucosa. These cells are targets for HIV entry and replication. Prophylactic HSV-2 vaccines (to prevent infection) and therapeutic vaccines (to modify or treat existing infections) are currently under development. By preventing or modifying infection, an effective HSV-2 vaccine could limit HSV-associated genital mucosal inflammation and thus HIV risk. However, a vaccine might have competing effects on HIV risk depending on its mechanism of action and cell populations generated in the genital mucosa. In this article, we review biologic interactions between HSV-2 and HIV-1, consider HSV-2 vaccine development in the context of HIV risk, and discuss implications and research needs for future HSV vaccine development.     

Safety and immunogenicity of long HSV-2 peptides complexed with rhHsc70 in HSV-2 seropositive persons

HSV-2, the primary causative agent of genital herpes, establishes latency in sensory ganglia and reactivates causing recurrent lesions and viral shedding. Induction or expansion of CD4⁺ and CD8⁺ T cell responses are expected to be important for a successful therapeutic vaccine against HSV-2. A candidate vaccine consisting of 32 synthetic 35mer HSV-2 peptides non-covalently complexed with recombinant human Hsc70 protein (named HerpV, formerly AG-707) was tested for safety and immunogenicity in a Phase I study. These peptides are derived from 22 HSV-2 proteins representative of all phases of viral replication. Thirty-five HSV-2 infected participants were randomized and treated in one of four groups: HerpV + QS-21 (saponin adjuvant), HerpV, QS-21, or vehicle. The vaccine was well tolerated and safe. All seven participants with evaluable samples who were administered HerpV with QS-21 demonstrated a statistically significant CD4⁺ T cell response to HSV-2 antigens, and the majority of such participants demonstrated a statistically significant CD8⁺ T cell response as well. To our knowledge, this is the first candidate vaccine against HSV-2 to demonstrate a broad CD4⁺ and CD8⁺ T cell response in HSV-2⁺ participants, and the first HSP-based vaccine to show immune responses against viral antigens in humans.     

A heat shock protein based polyvalent vaccine targeting HSV-2: CD4 and CD8 cellular immunity and protective efficacy

Efforts to develop a subunit vaccine against genital herpes have been hampered by lack of knowledge of the protective antigens of HSV-2, the causative agent of the disease. Vaccines based either on selected antigens or attenuated live virus approaches have not demonstrated meaningful clinical activity. We present here results of a therapeutic vaccine candidate, HerpV (formerly called AG-707), consisting of 32 HSV-2 peptides derived from 22 HSV-2 proteins, complexed non-covalently to the HSP70 chaperone and formulated with QS-21 saponin adjuvant. HerpV is observed to be immunogenic, generating CD4⁺ and CD8⁺ T cell responses in three mouse strains including HLA-A2 transgenic mice. Optimal T cell stimulation was dependent on the synergistic adjuvant properties of QS-21 with hsp70. The vaccine provided significant protection from viral challenge in a mouse prophylaxis model and showed signals of activity in a guinea pig therapeutic model of existing infection. Peripheral blood mononuclear cells from human HSV-2⁺ subjects also showed reactivity in vitro to a subset of individual peptides and to the pool of all 32 peptides. Recombinant human Hsc70 complexed with the 32 peptides also stimulated the expansion of CD8⁺ T cells from HSV-2⁺ subjects in vitro. These studies demonstrate that HerpV is a promising immunotherapy candidate for genital herpes, and provide a foundation for evaluating HerpV in human HSV-2⁺ subjects with the intent of eliciting CD4⁺ and CD8⁺ T cell responses to a broad array of viral antigens.     

Construction and properties of a herpes simplex virus 2 dl5-29 vaccine candidate strain encoding an HSV-1 virion host shutoff protein

The replication-defective herpes simplex virus 2 (HSV-2) dl5-29 mutant virus strain with deletions in the U_L5 and U_L29 genes has been shown to protect mice and guinea pigs against challenge with wild-type (wt) HSV-2 and to protect against ocular disease caused by HSV-1 infection. The dl5-29 strain is currently being prepared for clinical trials as a herpes vaccine candidate. As a possible approach to improve the efficacy of dl5-29 as a genital herpes vaccine, we replaced the U_L41 gene encoding the virion host shutoff function (vhs) with the U_L41 gene from HSV-1. While the HSV-2 U_L41 and HSV-1 U_L41 gene products have analogous functions, vhs-1 is 40-fold less active than vhs-2. Previously, it was shown that disruption of the U_L41 gene can increase the efficacy of dl5-29 as a vaccine against HSV-2. These properties led us to hypothesize that replacement of vhs-2 by vhs-1 would decrease cytopathic effects in infected host cells, allowing longer survival of antigen-presenting cells and induction of stronger immune responses. The new recombinant dl5-29-41.1 virus shows nearly the same immunogenicity and protection against HSV-2 challenge as the parental dl5-29 virus or a triply deleted mutant virus, dl5-29-41, in the murine model of infection, and grows to higher titers than the parental strain in complementing cells, which is important for GMP production. The results have implications for the design of future HSV-2 vaccine candidates and mechanisms of induction of protective immunity against genital herpes.     

Status of vaccine research and development of vaccines for herpes simplex virus

Herpes simplex virus type-1 (HSV-1) and -2 (HSV-2) are highly prevalent global pathogens which commonly cause recurrent oral and genital ulcerations. Less common but more serious complications include meningitis, encephalitis, neonatal infection, and keratitis. HSV-2 infection is a significant driver of the HIV epidemic, increasing the risk of HIV acquisition 3 fold. As current control strategies for genital HSV-2 infection, including antiviral therapy and condom use, are only partially effective, vaccines will be required to reduce infection. Both preventive and therapeutic vaccines for HSV-2 are being pursued and are in various stages of development. We will provide an overview of efforts to develop HSV-2 vaccines, including a discussion of the clinical need for an HSV vaccine, and status of research and development with an emphasis on recent insights from trials of vaccine candidates in clinical testing. In addition, we will touch upon aspects of HSV vaccine development relevant to low and middle income countries.     

Design of a multi-epitope protein vaccine against herpes simplex virus,human papillomavirus and Chlamydia trachomatis as the main causes of sexually transmitted diseases

Sexually transmitted diseases (STDs) have a profound effect on reproductivity and sexual health worldwide. According to world health organization (WHO) 375 million new case of STD, including chlamydia trachomatis (chlamydia), Neisseria gonorrhoeae, HSV, HPV has been reported in 2016. More than 30 diverse pathogenesis have identified to be transmitted through sexual intercourse. Of these, viral infections (hepatitis B, herpes simplex virus (HSV or herpes), HIV, and human papillomavirus (HPV) are incurable. However, symptoms caused by the incurable viral infections can be alleviated through treatment. Antimicrobial resistance (AMR) of sexually transmitted infections (STIs) to antibiotics has increased recent years, in this regard, vaccination is proposed as an important strategy for prevention or treatment of STDs. Vaccine against HPV 16 and 18 suggests a new approach for controlling STDs but until now, there is no prophylactic or therapeutic vaccine have been approved for HSV-2 and Chlamydia trachomatis (CT); in this reason, developing an efficient vaccine is inevitable. Recently, different combinatorial forms of subunit vaccines against two or three type of bacteria have been designed.In this study, to design a combinatorial vaccine against HSV, CT, and HPV, the E7 and L2 from HPV, glycoprotein D from HSV-2 and ompA from CT were selected as final antigens. Afterward, the immunodominant helper T lymphocytes (HTLs) and cytolytic T lymphocytes (CTLs) epitopes were chosen from aforesaid antigens. P30 (tetanus toxoid epitope) as universal T-helper were also added to the vaccine. Moreover, flagellin D_1/D₀ as TLR5 agonist and the RS09 as a TLR4 ligand were incorporated to N and C-terminals of peptide vaccine, respectively. Finally, all selected parts were fused together by appropriate linkers to enhance vaccine efficiency. The physicochemical, structural, and immunological properties of the designed vaccine protein were assessed. To achieve the best 3D model of the protein vaccine, modeling, refinement, and validation of modeled structures were also done. Docking evaluation demonstrated suitable interaction between the vaccine and TLR5. Moreover, molecular dynamics (MD) studies showed an appropriate and stable structure of protein and TLR5. Based on immunoinformatic analysis, our vaccine candidate could potentially incite humoral and cellular immunities, which are critical for protection against HPV, HSV-2, and chlamydia trachomatis. It should be noted that, experimental studies are needed to confirm the efficacy of the designed vaccine.     

Evaluation of murine bone marrow-derived dendritic cells loaded with inactivated virus as a vaccine against Japanese encephalitis virus

Japanese encephalitis (JE) is a serious infectious disease in southern and eastern Asia. Design and development of safer and more efficacious vaccines against Japanese encephalitis virus (JEV) is a high-priority target in the world. Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) playing a central and unique role in the generation of primary T-cell responses, and are considered attractive “live adjuvants” for vaccination and immunotherapy against cancer and infectious diseases. In this study, mouse bone marrow-derived dendritic cells (bmDCs were generated and stimulated with inactivated JEV in vitro. BALB/c mice were immunized with stimulated bmDCs and then challenged with JEV wild-type strain. The neutralization antibody, interferon gamma (IFN-γ), tumor necrosis factors alpha (TNF-α) or interleukin-6 (IL-6), and virus-specific CD8+ cytotoxic T-lymphocyte (CTL) levels, as well as survival rates, were analyzed and compared with inactivated vaccine and DCs control groups. The results demonstrated that intravenous (i.v.) injection of 2 × 10⁵ JEV-pulsed bmDCs into each mouse produced notable levels of JEV-specific neutralizing antibodies and higher levels of CD8+ CTL, IFN-γ and TNF-α compared with JEV-inactivated vaccine. Furthermore, stimulated bmDCs could elicit a highly protective efficacy (90%) against JEV challenge. It suggests that stimulated bmDCs can be considered as an attractive “live adjuvant” for vaccination against JEV infection.     

Laboratory investigations of herpes simplex virus-1 and -2 clinical samples in Korea

ObjectivesHerpes simplex virus (HSV) infections have been reported in 60% to 95% of the adult population worldwide, making them one of the most common infectious conditions globally. HSV-1 and HSV-2 cause severe disease in immunocompromised patients. Therefore, the aim of this study was to provide information that could be used to reduce the incidence of genital herpes caused by HSV-1 and HSV-2.MethodsFrom September 2018 to December 2020, 59,381 specimens were collected from outpatients across primary and secondary hospitals in Korea who requested U2Bio (Korea) to conduct molecular biological testing of their samples for sexually transmitted infections. In this study, the positivity rates of HSV-1 and HSV-2 were analyzed according to age, sex, and specimen type.ResultsIn the age-specific analysis of HSV-1, the highest positivity rate (0.58%) was observed in patients under 19 years of age, whereas the lowest positivity rate (0.08%) was observed in patients aged over 70 years. In the age-specific analysis of HSV-2, the highest positivity rate (2.53%) was likewise observed in patients under 19 years of age.ConclusionOur study identified differences in the infection rates of HSV-1 and HSV-2 depending on patients’ sex and age. These differences will be useful for improving disease prevention and control measures for HSV-1 and HSV-2.     

Vaccine prevents genital herpes in subgroup of women

The herpes simplex virus (HSV) type-2 vaccine studied here prevented genital herpes only in women who were seronegative for HSV-1 and HSV-2 at baseline. Ten of these women would need to be vaccinated to prevent 1 case of genital herpes. The vaccine did not prevent infection with HSV-2 in these women. It did not prevent genital herpes in women with other HSV serologic status or in men. The usefulness of this vaccine is limited by the small subgroup in which it is efficacious. Determining which women fall into this subgroup could prove costly. It is possible that asymptomatic infected persons may spread HSV more readily. Emphasis on the use of condoms and antiviral agents should still be the first line in preventing the spread of genital herpes.     

A multistage-polyepitope vaccine protects against Mycobacterium tuberculosis infection in HLA-DR3 transgenic mice

Mycobacterium tuberculosis (Mtb) is responsible for almost 2 million deaths annually. BCG, currently the only TB vaccine, induces variable protection and does not protect against reactivation of latent TB. Thus, efficient vaccines to supplement BCG are required urgently. Since Mtb's proteome differs qualitatively and quantitatively during bacterial replication stages from that expressed during dormancy, improved TB vaccines should drive immune responses to Mtb antigens expressed during multiple stages of infection. Consequently, such “multistage” vaccines should be composed of (immunodominant) antigens expressed during different phases of Mtb infection. As a concept multistage vaccine, we constructed a polyepitope by fusing five HLA-DR3-restricted T-cell epitopes derived from different Mtb proteins either expressed highly by replicating bacteria (Ag85B, hsp65, 19 kDa lipoprotein), or abundantly expressed by dormant bacilli and recognized preferentially by TST⁺ individuals (hsp16, Rv1733c). PBMC of HLA-DR3⁺ but not HLA-DR3⁻ cured TB patients and TST⁺ individuals responded well to the multistage-polyepitope in vitro. The in vivo immunogenicity and protective efficacy of the multistage-polyepitope were analyzed using HLA-DR3 transgenic mice lacking endogenous murine class II as a model. Immunization with the multistage-polyepitope adjuvanted with CpG generated high IgG levels as well as polyfunctional CD4⁺ T-cells producing IFN-γ, TNF and IL-2, specific for these HLA-DR3-restricted epitopes. Importantly, multistage-polyepitope immunization reduced the number of bacilli in the lungs after Mtb challenge when administered as prophylactic vaccine. Given the extensive repertoire of potential Mtb antigens available for immune recognition, the data of our model demonstrate the potential of multistage-polyepitope vaccines to protect against TB.     

Hypothetical vaccination of the Dutch population with a herpes simplex virus vaccine: estimation of the profitability using a demographic projection model

The apparently increasing evidence of herpes simplex virus infections of the genital tract has focused attention on preventing the infection by vaccination. Herpes genitalis is not, however, the most quantitatively important clinical manifestation of herpes simplex virus infections. Because 41% of the hospitalized patients are younger than 20 years, vaccination of birth cohorts would be more favourable. In this paper the financial benefits of a hypothetical herpes simplex virus vaccination were calculated with the use of a population projection model. For the Netherlands, if the price of the hypothetical herpes simplex virus vaccine equals the cost price of the mumps component of the combined mumps-measles-rubella vaccine, the herpes vaccine would be profitable within 8 years.     

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.