Impact on antitumor response using a new adjuvant preparation as a component of a human papillomavirus type 16 therapeutic vaccine candidate.

Cervical cancer is a global public health problem and human papillomavirus (HPV) 16 accounts for approximately 50% of cases worldwide. Although there are several types of HPV therapeutic vaccines in clinical research, there are currently not approved for use in humans. We developed the fusion protein LALF32-51-E7 (hereafter denominated CIGB550-E7) defined by a cell-penetrating peptide linked to an E7 mutein for the treatment of HPV16-associated tumors. We have demonstrated previously the benefit on antitumor response induced by the immunization with CIGB550-E7 admixed with very small size proteoliposomes (VSSP) adjuvant compared with the adjuvant-free immunization. In this study, we obtained a similar antitumor response in mice immunized with CIGB550-E7 admixed with the new adjuvant sVSSP that does not contain any animal-derived product. Also, the immunization with the above mentioned vaccine preparation induced a cell-mediated immune response. Our results are encouraging for the future clinical trials with the vaccine candidate CIGB550-E7+sVSSP.

Growth hormone releasing peptide-6 enhanced antibody titers against subunit antigens in mice (BALB/c), tilapia (Oreochromis niloticus) and African catfish (Clarias gariepinus).

Modern subunit vaccines have excellent safety profiles and improved tolerability, but do not elicit strong immune responses without the addition of adjuvants. Developing a safe and affective adjuvant remains a challenge for peptide-based vaccine design. Growth Hormone Releasing Peptide-6 (GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand, ghrelin. In the present study, we evaluated the ability of GHRP-6 to enhance the humoral immune response against co-injected antigens in mice, tilapia and African catfish. This peptide was able to increase the antigen-specific antibody response using heterologous proteins and peptides as antigens, which were also formulated in "water in oil" emulsions (Freund and Montanide). As long as we know there is no previous report describing any ghrelin analogous as molecular immunomodulator stimulating a humoral immune response. Further studies will be conducted to evaluate the functionality of this humoral immune response in challenge trials.

LALF32-51-E7 therapeutic vaccine induces antitumor immunity against human papillomavirus type 16 E7-expressing murine tumor metastases in the lungs.

One important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported the significant antitumor effect induced by the immunization with our human papillomavirus therapeutic protein-based vaccine (LALF32-51-E7) without adjuvant and admixed with clinically relevant adjuvants in the subcutaneous TC-1 tumor challenge model. In the present study, we evaluated the efficacy of the above mentioned vaccine formulations in controlling the hematogenous spread of TC-1 tumor cells using a more tumourigenic clone named TC-1* and other intravenous injection site less stressful than the tail vein. We generated a lung metastasis model by injecting TC-1* cells into the retro-orbital venous sinus and this is the first study describing it. Also, this is the first study that demonstrates the efficacy of the immunization with LALF32-51-E7 without adjuvant and admixed with VSSP or Al(OH)3 in controlling metastatic tumors increasing the survival of the mice. Our TC-1 lung metastasis model can be used to test the efficacy of other immunotherapeutic strategies based on E6/E7 antigens.

A Tetravalent Formulation Based on Recombinant Nucleocapsid-like Particles from Dengue Viruses Induces a Functional Immune Response in Mice and Monkeys.

Despite the considerable effort that has been invested in elucidating the mechanisms of protection and immunopathogenesis associated with dengue virus infections, a reliable correlate of protection against the disease remains to be found. Neutralizing Abs, long considered the prime component of a protective response, can exacerbate disease severity when present at subprotective levels, and a growing body of data is challenging the notion that their titers are positively correlated with disease protection. Consequently, the protective role of cell-mediated immunity in the control of dengue infections has begun to be studied. Although earlier research implicated cellular immunity in dengue immunopathogenesis, a wealth of newer data demonstrated that multifunctional CD8+ T cell responses are instrumental for avoiding the more severe manifestations of dengue disease. In this article, we describe a new tetravalent vaccine candidate based on recombinant dengue virus capsid proteins, efficiently produced in Escherichia coli and purified using a single ion-exchange chromatography step. After aggregation to form nucleocapsid-like particles upon incubation with an oligodeoxynucleotide containing immunostimulatory CpG motifs, these Ags induce, in mice and monkeys, an IFN-γ-secreting cell response that significantly reduces viral load after challenge without the contribution of antiviral Abs. Therefore, this new vaccine candidate may not carry the risk for disease enhancement associated with Ab-based formulations.

Dengue encephalitis-associated immunopathology in the mouse model: Implications for vaccine developers and antigens inducer of cellular immune response.

Despite the many efforts made by the scientific community in the development of vaccine candidates against dengue virus (DENV), no vaccine has been licensed up to date. Although the immunopathogenesis associated to the disease is a key factor to take into account by vaccine developers, the lack of animal models that reproduce the clinical signs of the disease has hampered the vaccine progress. Non-human primates support viral replication, but they are very expensive and do not show signs of disease. Immunocompromised mice develop viremia and some signs of the disease; however, they are not valuable for vaccine testing. Nowadays, immunocompetent mice are the most used model to evaluate the immunogenicity of vaccine candidates. These animals are resistant to DENV infection; therefore, the intracranial inoculation with neuroadapted virus, which provokes viral encephalitis, represents an alternative to evaluate the protective capacity of vaccine candidates. Previous results have demonstrated the crucial role of cellular immune response in the protection induced by the virus and vaccine candidates in this mouse encephalitis model. However, in the present work we are proposing that the magnitude of the cell-mediated immunity and the inflammatory response generated by the vaccine can modulate the survival rate after viral challenge. We observed that the intracranial challenge of naïve mice with DENV-2 induces the recruitment of immune cells that contribute to the reduction of viral load, but does not increase the survival rate. On the contrary, animals treated with cyclophosphamide, an immunosuppressive drug that affects proliferating lymphocytes, had a higher viral load but a better survival rate than untreated animals. These results suggest that the immune system is playing an immunopathogenic role in this model and the survival rate may not be a suitable endpoint in the evaluation of vaccine candidates based on antigens that induce a strong cellular immune response.

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.

A vaccine formulation consisting of nucleocapsid-like particles from Dengue-2 and the fusion protein P64k-domain III from Dengue-1 induces a protective immune response against the homologous serotypes in mice.

In previous studies we reported the cloning, expression and purification of the capsid protein from Dengue-2 virus. Subsequently, we described an in vitro-assembly process for the capsid protein, which resulted in nucleocapsid-like particles (recNLPs) that induced functional cell-mediated immunity and protection in mice. Moreover, our group reported the evaluation in non-human primates of the fusion protein P64k-domain III from Dengue-1 (PD10). This protein proved to be immunogenic and protective when Freund's adjuvant, but not alum, was used. Based on the previously demonstrated capacity of recNLPs to potentiate the immunogenicity of heterologous proteins, in this study we assess the immune response elicited by the formulation PD10-recNLPs-alum and its protective capacity against Dengue-1 and Dengue-2 virus. As expected, the humoral immune response was mainly directed against Dengue-1, while high levels of IFN-γ secretion were detected after stimulation with Dengue-1 and Dengue-2. Consistently, animals immunized with the bivalent formulation were significantly protected against challenge with either Dengue serotype. In conclusion, this report describes a novel formulation based on recombinant proteins and alum, which is protective against Dengue-1 and Dengue-2 in mice.

CIGB-247: a VEGF-based therapeutic vaccine that reduces experimental and spontaneous lung metastasis of C57Bl/6 and BALB/c mouse tumors.

CIGB-247 is a novel cancer therapeutic vaccine that uses a mutated form of human VEGF as antigen. Being metastatic disease the most dramatic factor of tumor biology affecting patient survival and cure, preclinical evaluation of the impact of CIGB-247 vaccination on experimental metastasis mouse models is highly relevant, and constitutes the focus of this work. CIGB-247 was administered in a weekly schedule known to effectively reduce primary tumor growth. The vaccine was tested in experimental and spontaneous metastasis models of colon (CT26), lung (3LL-D122) and breast (F3II) carcinomas growing in C57Bl/6 or BALB/c mice. Primary tumor growth parameters, metastatic counts, and/or animal survival were recorded. Histology and specific humoral and cellular responses to the vaccine were evaluated. As compared to control groups, CIGB-247 vaccination significantly reduced the number and size of metastatic tumor foci in lungs after intravenous inoculation of CT26 and 3LL-D122 tumor cells. Spontaneous lung dissemination from 3LL-D122 and F3II breast tumor cells implanted in the footpad, or subcutaneously, was also reduced by immunization with CIGB-247. The vaccine elicited in both mouse strains antibodies specific for human and murine VEGF that effectively blocked the interaction of VEGF with VEGF receptor 2. Differing from other experimental reports that describe the use of VEGF for active tumor immunotherapy, CIGB-247 elicited a specific cellular response, measured both by a DTH increment and the induction of spleen cells cytotoxic to syngeneic tumor cells producing murine VEGF. In summary our results reinforce the potential of CIGB-247 vaccination to reduce both tumor growth and the number and size of tumor metastasis in lungs, the latter both after direct inoculations of cells in the blood stream, or as part of primary tumor progression in immunocompetent mice.

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.

Nucleocapsid-like particles of dengue-2 virus enhance the immune response against a recombinant protein of dengue-4 virus.

In this study, we evaluate in mice a novel formulation containing nucleocapsid-like particles of dengue-2 virus (recNLP) co-immunized with a chimeric protein composed of the dengue-4 envelope domain III fused twice within the meningococcal P64k protein of Neisseria meningitidis (PD24). The animals receiving the PD24-recNLP mixture showed the highest levels of antiviral antibodies. Similar results were obtained for IFNγ secretion levels, indicating a functional Th1 cellular response. Consistently, the percentage of mice surviving after viral challenge was significantly higher for those immunized with the mixture than for those inoculated with PD24 protein alone. In addition, in vivo depletion experiments demonstrated the decisive role of CD4(+) and CD8(+) cells in the protection conferred by immunization with PD24-recNLP. In conclusion, this report demonstrates for the first time the adjuvant capacity of dengue-2 virus recNLP. Additionally, the evidence presented highlights the potential of these particles for enhancing the immune response against heterologous recombinant proteins.

The cellular immune response plays an important role in protecting against dengue virus in the mouse encephalitis model.

For several years, researchers have known that the generation of neutralizing antibodies is a prerequisite for attaining adequate protection against dengue virus. Nevertheless, the cellular immune response is the principal arm of the adaptive immune system against non-cytopathic viruses such as dengue, as once the virus enters into the cell it is necessary to destroy it to eliminate the virus. To define the role of the cellular immune response in the protection against dengue, we selected the mouse encephalitis model. Mice were immunized with a single dose of infective dengue 2 virus and different markers of both branches of the induced adaptive immunity were measured. Animals elicited a broad antibody response against the four dengue virus serotypes, but neutralizing activity was only detected against the homologous serotype. On the other hand, the splenocytes of the infected animals strongly proliferated after in vitro stimulation with the homologous virus, and specifically the CD8 T-cell subset was responsible for the secretion of the cytokine IFN-gamma. Finally, to define the role of T cells in in vivo protection, groups of animals were inoculated with the depleting monoclonal antibodies anti-CD4 or anti-CD8. Only depletion with anti-CD8 decreased to 50% the level of protection reached in the non-depleted mice. The present work constitutes the first report defining the role of the cellular immune response in protection against dengue virus in the mouse model.

Dengue-4 envelope domain III fused twice within the meningococcal P64k protein carrier induces partial protection in mice.

A vaccine against dengue virus must be able to induce an effective and equivalent immune response to the four viral serotypes; however, some studies have revealed that DEN4 (dengue-virus serotype 4) induces a weaker immune response than the others in quadrivalent (tetravalent') formulations. We have previously reported the protective capacity, in a viral encephalitis murine model, of fusion protein P64k-envelope domain III of DEN1, DEN2 and DEN3. We also reported that the P64k protein can be used as a carrier in two different positions: the insertion following the first 45 amino acids and the fusion at the C-terminus. Considering the low immunogenicity described for DEN4, in the present study we obtained a novel chimaeric protein by inserting two dengue-4 envelope domains III in both sites of P64k (PD24), and hence increasing the presence of the virus in the final construct. After expression in Escherichia coli and semipurification, the protein exhibited a pattern of high molecular mass and was well recognized by human and murine polyclonal antibodies. The protein was finally evaluated in mice, Al(OH)(3) being employed as the adjuvant. Even though the animals exhibited low levels of antiviral antibodies, the recombinant protein induced significant protection against lethal challenge with dengue-4 virus.

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.