Alexandre Krendelchtchikov

Using Multivalent Adenoviral Vectors for HIV Vaccination

Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the “antigen capsid-incorporation” strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His6 within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His6 epitope-specific humoral immune response.

Neural stem cell-derived exosomes mediate viral entry

Background Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells. Methods To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5) for the proof-of-principle study. Results Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR)-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4), which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry. Conclusion Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines.

A recombinant adenovirus-based vector elicits a specific humoral immune response against the V3 loop of HIV-1 gp120 in mice through the "Antigen Capsid-Incorporation" strategy.

BackgroundDue to potential advantages, human adenoviral vectors have been evaluated pre-clinically as recombinant vaccine vectors against several cancers and infectious diseases, including human immunodeficiency virus (HIV) infection. The V3 loop of HIV-1 glycoprotein 120 (gp120) contains important neutralizing epitopes and plays key roles in HIV entry and infectivity.MethodsIn order to investigate the humoral immune response development against portions of the V3 loop, we sought to generate four versions of adenovirus (Ad)-based V3 vectors by incorporating four different antigen inserts into the hypervariable region 1 (HVR1) of human adenovirus type 5 (hAd5) hexon. The strategy whereby antigens are incorporated within the adenovirus capsid is known as the “Antigen Capsid-Incorporation” strategy.ResultsOf the four recombinant vectors, Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 had the capability to present heterologous antigens on capsid surface, while maintaining low viral particle to infectious particle (VP/IP) ratios. The VP/IP ratios indicated both high viability and stability of these two vectors, as well as the possibility that V3 epitopes on these two vectors could be presented to immune system. Furthermore, both Ad-HVR1-lgs-His6-V3 and Ad-HVR1-long-V3 could, to some extent escape the neutralization by anti-adenovirus polyclonal antibody (PAb), but rather not the immunity by anti-gp120 (902) monoclonal antibody (MAb). The neutralization assay together with the whole virus enzyme-linked immunosorbent assay (ELISA) suggested that these two vectors could present V3 epitopes similar to the natural V3 presence in native HIV virions. However, subsequent mice immunizations clearly showed that only Ad-HVR1-lgs-His6-V3 elicited strong humoral immune response against V3. Isotype ELISAs identified IgG2a and IgG2b as the dominant IgG isotypes, while IgG1 comprised the minority.ConclusionsOur findings demonstrated that human adenovirus (hAd) vectors which present HIV antigen via the “Antigen Capsid-Incorporation” strategy could successfully elicit antigen-specific humoral immune responses, which could potentially open an avenue for the development of Ad-based HIV V3 vaccines.

Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

Exosomes, 30–200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies.

Differential binding of the HIV-1 envelope to phosphatidylserine receptors

Prior work has shown that the HIV-1 envelope of the human immunodeficiency virus (HIV) interacts directly with T-cell immunoglobulin mucin (TIM) family proteins. Herein, we demonstrate that HIV-1 envelope glycoproteins from varying HIV-1 clades bind differentially to TIM proteins and functionally similar proteins acting as phosphatidylserine (PtdSer) receptors. Using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) technology, we show that lysate containing HIV-1 envelope and recombinant HIV-1 envelope glycoproteins bind TIM-4 and advanced glycosylation end product-specific receptor (AGER). The complex binding of HIV-1 UG21 gp140 to TIM-4 or AGER suggests a biphasic interaction with these proteins.

Epitope Capsid-Incorporation: New Effective Approach for Vaccine Development for Chagas Disease

Background: Previously we reported that a hexon-modified adenovirus (Ad) vector containing the invasive neutralizing epitope of Trypanosoma cruzi (T. cruzi) trypomastigote gp83 (Ad5-gp83) provided immunoprotection against T. cruzi infection. The purpose of this work was to design an improved vaccine for T. cruzi using a novel epitope capsid incorporation strategy. Thus, we evaluated the immunoprotection raised by co-immunization with Ad5-gp83 and an Ad vector containing an epitope (ASP-M) of the T. cruzi amastigote surface protein 2. Methods: Protein IX (pIX)-modified Ad vector (Ad5-pIX-ASP-M) was generated, characterized, and validated. C3H/He mice were immunized with Ad5-pIX-ASP-M and Ad5-gp83 and the cell-mediated responses were evaluated by enzyme-linked immunospot (ELISPOT) assay and intracellular staining. Immunized mice were challenged with T. cruzi to evaluate the vaccine efficacy. Results: Our findings indicate that Ad5-pIX-ASP-M was viable. Specific CD8+ T-cell mediated responses prior to the challenge show an increase in IFNγ and TNFα production. A single immunization with Ad5-pIX-ASP-M provided protection from T. cruzi infection, but co-immunizations with Ad5-pIX-ASP-M and Ad5-gp83 provided a higher immunoprotection and increased survival rate of mice. Conclusions: Overall, these results suggest that the combination of gp83 and ASP-M specific epitopes onto the capsid-incorporated adenoviruses would provide superior protection against Chagas disease as compared with Ad5-gp83 alone.

A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease.

Development of an Ad5H3 Chimera Using the “Antigen Capsid-Incorporation” Strategy for an Alternative Vaccination Approach

Background: Adenovirus type 5 (Ad5) achieved success as a conventional transgene vaccine vector in preclinical trials, however; achieved poor efficiency in some of the clinical trials, due to the major hurdle associated with Ad5 pre-existing immunity (PEI) in the majority of the human population. Objective: We sought to generate Ad5-based chimeras to assess their capabilities to bypass this bottleneck and to induce antigen-specific humoral immune response. Methods: A His6 tag was incorporated into the hypervariable region 2 (HVR2) of hexon3 (H3) capsid protein using the “Antigen Capsid-Incorporation” strategy. This lead to the construction of a viral chimera, Ad5H3-HVR2-His. Ad5H3 was generated previously by substituting the hexon of Ad5 (hexon5) with the hexon from adenovirus type 3 (Ad3). Results: His6 was presented on the viral capsid surface and recognized by a His6 antibody. An in vitro neutralization assay with Ad5 sera indicated the ability of Ad5 chimeras to partially escape Ad5 immunity. Immunization with Ad5H3-HVR2-His generated significant humoral response to the incorporated tagged peptide, when compared to the immunizations with controls. Conclusion: Based on our in vitro studies the data suggested that Ad5H3 as a novel chimeric vaccine platform yields the possibility to escape Ad5 neutralization, and the potential to generate robust humoral immunity against incorporated antigens using the “Antigen Capsid-Incorporation” strategy.

Viral Vectors for Vaccine Development

Recombinant vectors can be used to deliver antigens and to stimulate immune responses in humans. Viral vectors possess various intrinsic properties which may lend to advantages and disadvantages for usage for a given therapeutic application [reviewed by Larocca and Schlom] [1]. The safety and flexibility of recombinant viral vectors have lead to their usage in gene therapy, virotherapy, and vaccine applications. In this chapter, we will discuss the utility and importance of recombinant vectors as vaccine agents. This chapter will highlight some of the uses of recombinant viral vectors for therapeutic vaccines; and will mostly focus on the application of a range of recombinant viral vectors for prophylactic vaccines against infectious agents. More specifically, this chapter will focus in depth on the use of recombinant adenovirus (Ad) for vaccine development against infectious agents.

Tetraspanin blockage reduces exosome-mediated HIV-1 entry

HIV-1 is one of the most studied retroviruses. The role of exosomes in HIV-1 entry and pathogenesis are beginning to be appreciated. Exosomes can incorporate host proteins that are also contained in viruses (e.g., tetraspanins).