A Nasal Vaccine Candidate, Containing Three Antigenic Regions from SARS-CoV-2, to Induce a Broader Response.

A chimeric protein, formed by two fragments of the conserved nucleocapsid (N) and S2 proteins from SARS-CoV-2, was obtained as a recombinant construct in Escherichia coli. The N fragment belongs to the C-terminal domain whereas the S2 fragment spans the fibre structure in the post-fusion conformation of the spike protein. The resultant protein, named S2NDH, was able to form spherical particles of 10 nm, which forms aggregates upon mixture with the CpG ODN-39M. Both preparations were recognized by positive COVID-19 human sera. The S2NDH + ODN-39M formulation administered by the intranasal route resulted highly immunogenic in Balb/c mice. It induced cross-reactive anti-N humoral immunity in both sera and bronchoalveolar fluids, under a Th1 pattern. The cell-mediated immunity (CMI) was also broad, with positive response even against the N protein of SARS-CoV-1. However, neither neutralizing antibodies (NAb) nor CMI against the S2 region were obtained. As alternative, the RBD protein was included in the formulation as inducer of NAb. Upon evaluation in mice by the intranasal route, a clear adjuvant effect was detected for the S2NDH + ODN-39M preparation over RBD. High levels of NAb were induced against SARS-CoV-2 and SARS-CoV-1. The bivalent formulation S2NDH + ODN-39M + RBD, administered by the intranasal route, constitutes an attractive proposal as booster vaccine of sarbecovirus scope.

The Nucleocapsid Protein of SARS-CoV-2, Combined with ODN-39M, Is a Potential Component for an Intranasal Bivalent Vaccine with Broader Functionality.

Despite the rapid development of vaccines against COVID-19, they have important limitations, such as safety issues, the scope of their efficacy, and the induction of mucosal immunity. The present study proposes a potential component for a new generation of vaccines. The recombinant nucleocapsid (N) protein from the SARS-CoV-2 Delta variant was combined with the ODN-39M, a synthetic 39 mer unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), used as an adjuvant. The evaluation of its immunogenicity in Balb/C mice revealed that only administration by intranasal route induced a systemic cross-reactive, cell-mediated immunity (CMI). In turn, this combination was able to induce anti-N IgA in the lungs, which, along with the specific IgG in sera and CMI in the spleen, was cross-reactive against the nucleocapsid protein of SARS-CoV-1. Furthermore, the nasal administration of the N + ODN-39M preparation, combined with RBD Delta protein, enhanced the local and systemic immune response against RBD, with a neutralizing capacity. Results make the N + ODN-39M preparation a suitable component for a future intranasal vaccine with broader functionality against Sarbecoviruses.

Broad humoral immunity generated in mice by a formulation composed of two antigens from the Delta variant of SARS-CoV-2.

Due to the rapid development of new variants of SARS-CoV-2 as well as the real threat of new coronavirus zoonosis events, the development of a preventive vaccine with a broader scope of functionality is highly desirable. Previously, we reported the functionality of a nasal formulation containing the nucleocapsid protein and the receptor-binding domain (RBD) of the spike protein of the Delta variant of SARS-CoV-2 combined with the ODN-39M adjuvant. This combination induced cross-reactive immunity in mucosal and systemic compartments at the sarbecovirus level. In the present study, we explored the magnitude of the immunity generated in BALB/c mice by the same formulation with alum added as an additional adjuvant, to enhance the humoral immunity against the two antigens. Animals were immunized with three doses of the bivalent formulation, administered by subcutaneous route. Humoral immunity was tested by ELISA, and the neutralizing capacity of the resulting antibodies (Abs) was evaluated using a surrogate test and a vesicular stomatitis virus (VSV) pseudovirus-based assay. Cell-mediated immunity was also investigated using an IFN-γ ELISpot assay. High levels of antibodies against both antigens (N and RBD) were obtained upon immunization. Anti-RBD Abs with neutralizing capacity reacted with the RBD of three SARS-CoV-2 variants tested, including Omicron. Abs recognizing the nucleocapsid proteins of SARS-CoV-1 and the SARS-CoV-2 Delta and Omicron variants were also detected. Taken together, these results suggest that this bivalent formulation could be an attractive component of a pancorona vaccine able to broaden the scope of humoral immunity against both antigens. This will be particularly important for the reinforcement of immunity in previously vaccinated and/or infected populations.

Cross-Reactive Profile Against Two Conserved Coronavirus Antigens in Sera from SARS-CoV-2 Hybrid and Vaccinated Immune Donors.

Since the beginning of the pandemic, the pre-existing immunity against SARS-CoV-2 has been postulated as one possible cause of asymptomatic infections. Later, various works reported that pre-existing immune response against the two structural conserved antigens: S2 subunit and the nucleocapsid protein, were associated to some level of asymptomatic profile in infected individuals. To explore the Ab background against these two antigens, in the context of vaccine-elicited and hybrid (natural infection plus vaccination induced) immunity of SARS-CoV-2, in this work, we tested sera from inactivated vaccine-immunized donors and from vaccinated and subsequent natural infected donors upon the Omicron variant wave in Guangdong province, China. Serum samples were collected from 27 COVID-19 convalescent, 25 SARS-CoV-2 vaccinated, and 10 negative donors. The IgG cross-reactivity response against these two antigens from another relevant human coronavirus (HCoV) was also evaluated. The findings indicate that IgG response against S2 and N protein was particularly higher in sera with hybrid immunity. The cross-reactive Abs were more significant against SARS-CoV-1, while a wide cross-reactivity was detected for N antigen for one human Alpha coronavirus HCoV-229E even in the negative control samples. The presence of cross-reactive Abs against the two conserved antigens N and S2, particularly in the context of hybrid immunity, could pave the way for future boosted vaccines carrying these conserved regions.

Evaluation of Cell-Penetrating Peptides as Mucosal Immune Enhancers for Nasal Vaccination.

Cell-penetrating peptides (CPPs) have been evaluated as enhancers in drug delivery, their addition in medical formulations favors drug absorption allowing obtaining the pharmacological effect with lower doses. In vaccine formulations their inclusion has been also explored with interesting results. Currently mucosal vaccination constitutes a promising alternative with the main advantage of inducing both systemic and mucosal immune responses, which are crucial for control tumors and infections at mucosal tissues. In the present work the nasal immune-enhancing effect of four CPPs was evaluated in Balb/c mice. Animals were intranasally immunized with CPP and the recombinant hepatitis B surface protein (HBsAg) as model antigen. The antibody response in sera and mucosal tissue was measured by ELISA. The IFN-γ secretion response at spleen was also evaluated by ELISPOT and ELISA. Among the CPPs studied one novel peptide stand out by its ability to potentiate the humoral and cellular immune response against the co-administered antigen. Considering that the use of mucosal routes is a promising strategy in vaccination, which are gaining special relevance nowadays in the development of novel candidates against SARS-CoV-2 and other potential emerging respiratory virus, the searching and development of safe mucosal adjuvants constitute a current need.

Implication of B23/NPM1 in Viral Infections, Potential Uses of B23/NPM1 Inhibitors as Antiviral Therapy.

BACKGROUND: B23/nucleophosmin (B23/NPM1) is an abundant multifunctional protein mainly located in the nucleolus but constantly shuttling between the nucleus and cytosol. As a consequence of its constitutive expression, intracellular dynamics and binding capacities, B23/NPM1 interacts with multiple cellular factors in different cellular compartments, but also with viral proteins from both DNA and RNA viruses. B23/NPM1 influences overall viral replication of viruses like HIV, HBV, HCV, HDV and HPV by playing functional roles in different stages of viral replication including nuclear import, viral genome transcription and assembly, as well as final particle formation. Of note, some virus modify the subcellular localization, stability and/or increases B23/NPM1 expression levels on target cells, probably to foster B23/NPM1 functions in their own replicative cycle. RESULTS: This review summarizes current knowledge concerning the interaction of B23/NPM1 with several viral proteins during relevant human infections. The opportunities and challenges of targeting this well-conserved host protein as a potentially new broad antiviral treatment are discussed in detail. Importantly, although initially conceived to treat cancer, a handful of B23/NPM1 inhibitors are currently available to test on viral infection models. CONCLUSION: As B23/NPM1 partakes in key steps of viral replication and some viral infections remain as unsolved medical needs, an appealing idea may be the expedite evaluation of B23/NPM1 inhibitors in viral infections. Furthermore, worth to be addressed is if the up-regulation of B23/NPM1 protein levels that follows persistent viral infections may be instrumental to the malignant transformation induced by virus like HBV and HCV.

Nasal route favors the induction of CD4+ T cell responses in the liver of HBV-carrier mice immunized with a recombinant hepatitis B surface- and core-based therapeutic vaccine.

Immunization routes and number of doses remain largely unexplored in therapeutic vaccination. The aim of the present work is to evaluate their impact on immune responses in naïve and hepatitis B virus (HBV)-carrier mouse models following immunization with a non-adjuvanted recombinant vaccine comprising the hepatitis B surface (HBsAg) and core (HBcAg) antigens. Mice were immunized either by intranasal (i.n.), subcutaneous (s.c.) or simultaneous (i.n. + s.c.) routes. Humoral immunity was detected in all the animal models with the induction of a potent antibody (Ab) response against HBcAg, which was stronger than the anti-HBs response. In the HBV-carrier mouse model, the anti-HBs response was predominantly subtype-specific and preferentially induced by the i.n. route. However, the Ab titers were not sufficient to clear the high concentration of HBsAg present in the sera of these mice. The i.n. route was the most efficacious at inducing cellular immune responses, in particular CD4+ T cells. In naïve mice, cellular responses in spleen were strong and mainly due to CD4+ T cells whereas the CD8+ T-cell response was low. In HBV-carrier mice, high frequencies of HBs-specific CD4+ T cells secreting interferon (IFN)-γ, interleukin (IL)-2 and tumor necrosis factor (TNF)-α were found in liver only after i.n. immunization. Increased frequencies of CD4+ T cells expressing the integrin CD49a in liver suggest a role of nasal route in the cellular homing process. Multiple dose schedules appear to be a prerequisite for protein-based immunization in order to overcome immunotolerance in HBV-carrier mice. These findings provide new avenues for further preclinical and clinical development.

Five-year Follow-up of Chronic Hepatitis B Patients Immunized by Nasal Route with the Therapeutic Vaccine HeberNasvac.

A novel therapeutic vaccine for chronic hepatitis B (CHB) treatment comprising the recombinant hepatitis B surface (HBsAg) and nucleocapsid (HBcAg) antigens has been developed. Preclinical and clinical trials (CT) evidenced safety and immunogenicity in animal models as well as in phases I, II, and III clinical trials. A phase I CT has conducted in Cuba in 6 CHB patients refractory or incomplete responders to α-IFN. Patients were immunized ten times every two weeks via. nasal spray, with 100 ug HBsAg and 100 ug HBcAg. Clinical efficacy was monitored by assessing the levels of hepatitis B virus deoxyribonucleic acid (HBV DNA), alanine aminotransferase (ALT), HBeAg, and anti-HBeAg seroconversion as well as by qualitative/ quantitative HBsAg serology during this period. After a 5 year follow-up,HBeAg loss was verified in the three HBeAg (+) patients, in two cases with seroconversion to anti-HBeAg. A reduction to undetectable viral load was observed in 5 out of 6 patients, and in two cases HBsAg seroconversion was also detected. ALT increases above the 2X upper limit of normal (ULN) were only detected in HBeAg (+) patients and associated with HBe antigen loss. All patients had stiffness levels below 7.8 KPa by Fibroscan assessment at the end of this period. Although only a few patients were enrolled in this study, it seems that HeberNasvac may maintain some of the therapeutic effects for a prolonged period. How to cite this article: Fernandez G, Sanchez AL, Jerez E, Anillo LE, Freyre F, Aguiar JA, Leon Y, Cinza Z, Diaz PA, Figueroa N, Muzio V, Nieto GG, Lobaina Y, Aguilar A, Penton E, Aguilar JC. Five-year Follow-up of Chronic Hepatitis B Patients Immunized by Nasal Route with the Therapeutic Vaccine HeberNasvac. Euroasian J Hepatogastroenterol, 2018;8(2):133-139.

Chronic hepatitis B: Immunological profile and current therapeutic vaccines in clinical trials.

More than 250million people worldwide are chronically infected with hepatitis B virus (CHB), and over half a million die each year due to CHB-associated liver complications such as cirrhosis and hepatocellular carcinoma. The translation of immunological knowledge about CHB into therapeutic strategies aiming to a sustainable hepatitis B virus (HBV) clearance has been challenging. In recent years, however, the understanding on the immune effectors required to overcome chronicity has notably increased thanks to preclinical and clinical research. Therapeutic vaccination may prove to be useful for treating CHB patients when coupled with current antiviral agents and other immunomodulatory strategies. This review summarizes current data and future perspectives on therapeutic vaccination. Other treatment alternatives that could be combined with vaccines for a complete cure from hepatitis B virus infection are also discussed.

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.

In vitro stimulation with HBV therapeutic vaccine candidate Nasvac activates B and T cells from chronic hepatitis B patients and healthy donors.

Hepatitis B virus (HBV) chronic infections remain a considerable health problem worldwide. The standard therapies have demonstrated limited efficacy, side effects or need life-long treatments. Nowadays therapeutic vaccination is a promising option. Recently, we developed a new vaccine formulation called Nasvac, based on the combination of surface and core antigens from HBV. Clinical trials already performed showed good efficacy in virus control. However, the exact mode of action of Nasvac formulation remains unclear. So far the functional impairment of DCs during persistent HBV infection is a controversial issue. On the other hand, it is known that B cells may function as antigen presenting cells (APC) activating T cells. The hepatitis B core antigen contained in Nasvac vaccine is able to bind and activate a high frequency of naive human B cells. In the present study the surface expression of activation and exhaustion markers on B cells and the subsequent activation of T cells after in vitro stimulation with Nasvac antigens were evaluated in chronic HBV patients and healthy donors. B- and T-cell phenotype and proliferation were assessed by flow cytometry. Our results indicate that in contrast to exhaustions markers B cell activation markers were increased on both study groups after Nasvac stimulation. A shift toward an activation phenotype was observed for both B and T cells. The present work suggests that B cells could act as efficient APCs for Nasvac antigens in humans, which might suggest the use of activated B cells as immunotherapeutic strategy for chronic hepatitis B.

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.

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.