Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation.

The development of therapeutic vaccines against chronic hepatitis B requires the capacity of the formulation to subvert a tolerated immune response as well as the evaluation of histopathological damage resulting from the treatment. In the present study, the dynamicity of induced immune response to hepatitis B surface antigen (HBsAg) was evaluated in transgenic mice that constitutively express the HBsAg gene (HBsAg-tg mice). After immunization with a vaccine candidate containing both surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV), the effect of vaccination on clearance of circulating HBsAg and the potential histological alterations were examined. Transgenic (tg) and non-transgenic (Ntg) mice were immunized by intranasal (IN) and subcutaneous (SC) routes simultaneously. A control group received phosphate-buffered saline (PBS) by IN route and aluminum by SC route. Positive responses, at both humoral and cellular levels, were obtained after five immunizations in HBsAg-tg mice. Such responses were delayed and of lower intensity in tg mice, compared to vaccinated Ntg mice. Serum IgG response was characterized by a similar IgG subclass pattern. Even when HBsAg-specific CD8+ T cell responses were clearly detectable by gamma-interferon ELISPOT assay, histopathological alterations were not detected in any organ, including the liver and kidneys. Our study demonstrated, that it is possible to subvert the immune tolerance against HBsAg in tg mice, opening a window for new studies to optimize the schedule, dose, and formulation to improve the immune response to the therapeutic vaccine candidate. These results can be considered a safety proof to support clinical developments for the formulation under study. HOW TO CITE THIS ARTICLE: Freyre FM, Blanco A, Trujillo H, Hernández D, García D, Alba JS, Lopez M, Merino N, Lobaina Y, Aguilar JC. Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation. Euroasian J Hepato-Gastroenterol 2016;6(1):25-30.

HBV genotypic variability in Cuba.

The genetic diversity of HBV in human population is often a reflection of its genetic admixture. The aim of this study was to explore the genotypic diversity of HBV in Cuba. The S genomic region of Cuban HBV isolates was sequenced and for selected isolates the complete genome or precore-core sequence was analyzed. The most frequent genotype was A (167/250, 67%), mainly A2 (149, 60%) but also A1 and one A4. A total of 77 isolates were classified as genotype D (31%), with co-circulation of several subgenotypes (56 D4, 2 D1, 5 D2, 7 D3/6 and 7 D7). Three isolates belonged to genotype E, two to H and one to B3. Complete genome sequence analysis of selected isolates confirmed the phylogenetic analysis performed with the S region. Mutations or polymorphisms in precore region were more common among genotype D compared to genotype A isolates. The HBV genotypic distribution in this Caribbean island correlates with the Y lineage genetic background of the population, where a European and African origin prevails. HBV genotypes E, B3 and H isolates might represent more recent introductions.

Two mucosal-parenteral schedules to coadminister a multiantigenic formulation against HIV-1 in Balb/c mice.

Previous studies showed that simultaneous immunization through the nasal (IN) and subcutaneous (SC) route of a multiantigenic formulation induced a Th1 anti-HIV humoral and cellular immune responses. The formulation was comprised of a recombinant protein of HIV-1 (named CR3; Cellular Response number 3) and the surface and nucleocapsid antigens of hepatitis B virus. This study asks whether four times simultaneous administration through the IN and SC routes (SC+IN) of the multiantigenic formulation induces a similar systemic and mucosal immune responses than two sequential IN priming and two SC boosting (2IN&2SC) inoculations in mice. To answer this question, we tested the same total dose of each antigen per animal in both schedules of inoculation. We found that SC+IN and 2IN&2SC coadministration induced comparable levels of CR3(HIV)-specific IFN-γ-secreting cells and CD8+ cells proliferation in the systemic compartment of animals. Consistent with these findings, a similar Th1 profile considering anti-CR3 IgG1:IGg2a ratio was observed. Additionally, the level of IgG antibodies and the frequency of seroconverting animals in vagina were not different. However, in the case of IgA antibodies the same parameters were significantly higher in the SC+IN group. We also found important level of HBsAg-specific antibodies in serum and vaginal washes.

The effect of the parenteral route of administration on the immune response to simultaneous nasal and parenteral immunizations using a new HBV therapeutic vaccine candidate.

Chronic hepatitis B is a major health problem, with more than 350 million people infected worldwide. Available therapies have limited efficacy and require long-term continuous and expensive treatments, which often lead to the selection of resistant viral variants and rarely eliminate the virus. Immunotherapies have been investigated as a promising new approach. Several vaccine formulations have been clinically tested in chronic patients, none of which have clearly demonstrated efficacy so far. In this study we evaluated a new vaccination strategy comprising the simultaneous co-administration by the nasal and parenteral routes of a multicomponent vaccine formulation in BALB/C and HBsAg-transgenic mice. The formulation under study contains the surface and nucleocapsid antigens of the HBV, and was co-administered by the nasal route and three parenteral routes. For parenteral administration we also evaluated the immunogenicity of the antigenic mixture with alum or without the adjuvant. The immune response was evaluated by ELISA and IFN-γ ELISPOT assays. Our results indicate that all variants generated a strong antibody response in the sera against both antigens, but differed in their capacity to induce cellular immune responses against the surface antigen. Mice immunized by the nasal and subcutaneous routes without alum generated the highest IFN-γ-secreting CD8+ T-cell response, and results in this transgenic mouse model showed that there is no need to include alum. In conclusion, our results indicate that the immunization routes have to be carefully selected before carrying out clinical trials to optimize the immune response and promote further clinical development.

Anti-HIV-1 and anti-HBV immune responses in mice after parenteral and nasal co-administration of a multiantigenic formulation.

The cell-mediated immune response to HIV-1 is an essential element of the mechanisms for viral replication control. Currently, most of the vaccine candidates in clinical trials were developed to stimulate HIV-1-specific CD8+ cytotoxic (CTL) and CD4+ T helper (Th) lymphocytes. We have been working on a novel approach to develop a vaccine formulation for HIV-1 using a recombinant multiepitopic protein (named CR3), which comprises CTL and Th epitope-rich regions of HIV-1 from several subtype B isolates, co-inoculated with the hepatitis B virus surface (HBsAg) and core (HBcAg) antigens of the hepatitis B virus (HBV) as adjuvant. According to our studies in mice, the nasal-subcutaneous co-administration of this multiantigenic formulation induces a strong Th1-biased specific response against CR3, CD8+ T cells in mice spleen and IFN-gamma-secreting cells in mesenteric lymph nodes. Cross-reactive p24-specific IFN-gamma-secreting cells in spleen were also detected. Moreover, Nef-specific antibodies were elicited in mice sera which might avoid the toxic effects of this antigen. However, a marginal anti-CR3 antibody response was elicited in vaginal mucosa. Additionally, we observed anti-HBsAg and anti-HBcAg cellular and humoral responses. In this regard, our multiantigenic formulation might provide immunity against HBV as an additional benefic considering the high HIV-1-HBV co-infection rate reported worldwide.

Coinoculation with hepatitis B surface and core antigen promotes a Th1 immune response to a multiepitopic protein of HIV-1.

It has been defined that strong and multispecific cellular immune responses correlate with a better prognosis during the course of chronic diseases. A cross-enhancing effect on the resulting immune response obtained by the coadministration of recombinant hepatitis B virus (HBV) surface and core Ag was recently observed. With the objective of studying the effect of such Ag on the immune response to coinoculated heterologous Ag and vice versa, several formulations containing the recombinant HBV Ag and a multiepitopic protein (CR3) composed by CTL and Th epitopes from HIV-1 were evaluated by s.c. and mucosal administration. Combinations of two and three Ag were evaluated for cellular and humoral immune responses. The results showed that the best Ag combination for nasal immunization was the mixture comprising the CR3 recombinant HIV protein and both HBV Ag. Similarly, it was also the best formulation for s.c. immunization in aluminium phosphate adjuvant. In conclusion, it is possible to induce a Th1 stimulation of the cellular immune response specific for a HIV-based recombinant protein by formulating this Ag with the recombinant HBV Ag.

Influence of aluminum-based adjuvant on the immune response to multiantigenic formulation.

Several adjuvants have been described and tested in humans. However, the aluminum-based adjuvants remain the most widely used component in vaccines today. Emerging data suggest that aluminum phosphate and aluminum hydroxide adjuvants do not promote a strong commitment to the helper T cell type 2 (Th2) pathway when they are coadministered with some Th1 adjuvants. In this regard, subtle differences between both aluminum-based adjuvants have been demonstrated. We have previously shown that subcutaneous immunization, in aluminum phosphate, of a mixture comprising the surface and core antigens of hepatitis B virus (HBV) and the multiepitopic protein CR3 of human immunodeficiency virus type 1 elicits a CR3-specific Th1 immune response. In these experiments, the antigens were adjuvated at the same time. As the final selection of the best adjuvant should be based on experimental evidence, we asked whether aluminum hydroxide allows a better Th1 immune deviation than aluminum phosphate. We also studied several ways to mix the antigens and the impact on CR3-specific interferon (IFN)-gamma secretion. Our findings indicate that aluminum hydroxide allows better Th1 immunodeviation than aluminum phosphate adjuvant for the mixture of HBV antigens and CR3. In addition, CR3-specific IFN-gamma secretion of the various formulations tested was the same irrespective of the order in which the antigens were combined.