Enhancement of subunit vaccine delivery with zinc-carnosine coordination polymer through the addition of mannan.

Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs' limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.

Immunogenicity of an adjuvanted broadly active influenza vaccine in immunocompromised and diverse populations.

Influenza virus outbreaks are a major burden worldwide each year. Current vaccination strategies are inadequate due to antigenic drift/shift of the virus and the elicitation of low immune responses. The use of computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) immunogens subvert the constantly mutating viruses; however, they are poorly immunogenic on their own. To increase the immunogenicity of subunit vaccines such as this, adjuvants can be delivered with the vaccine. For example, agonists of the stimulator of interferon genes (STING) have proven efficacy as vaccine adjuvants. However, their use in high-risk populations most vulnerable to influenza virus infection has not been closely examined. Here, we utilize a vaccine platform consisting of acetalated dextran microparticles loaded with COBRA HA and the STING agonist cyclic GMP-AMP. We examine the immunogenicity of this platform in mouse models of obesity, aging, and chemotherapy-induced immunosuppression. Further, we examine vaccine efficacy in collaborative cross mice, a genetically diverse population that mimics human genetic heterogeneity. Overall, this vaccine platform had variable efficacy in these populations supporting work to better tailor adjuvants to specific populations.

Comparative study of acetalated-dextran microparticle fabrication methods for a clinically translatable subunit-based influenza vaccine.

The most common influenza vaccines are inactivated viruses produced in chicken eggs, which is a time-consuming production method with variable efficacy due to mismatches of the vaccine strains to the dominant circulating strains. Subunit-based vaccines provide faster production times in comparison to the traditional egg-produced vaccines but often require the use of an adjuvant to elicit a highly protective immune response. However, the current FDA approved adjuvant for influenza vaccines (MF59) elicits a primarily helper T-cell type 2 (Th2)-biased humoral immune response. Adjuvants that can stimulate a Th1 cellular response are correlated to have more robust protection against influenza. The cyclic dinucleotide cGAMP has been shown to provide a potent Th1 response but requires the use of a delivery vehicle to best initiate its signalling pathway in the cytosol. Herein, acetalated dextran (Ace-DEX) was used as the polymer to fabricate microparticles (MPs) via double-emulsion, electrospray, and spray drying methods to encapsulate cGAMP. This study compared each fabrication method's ability to encapsulate and retain the hydrophilic adjuvant cGAMP. We compared their therapeutic efficacy to Addavax, an MF59-like adjuvant, and cGAMP Ace-DEX MPs provided a stronger Th1 response in vaccinated BALB/c mice. Furthermore, we compared Ace-DEX MPs to spray dried MPs composed from a commonly used polymer for drug delivery, poly(lactic-co-glycolic acid) (PLGA). We observed that all Ace-DEX MPs elicited similar humoral and cellular responses to the PLGA MPs. Overall, the results shown here indicate Ace-DEX can perform similarly to PLGA as a polymer for drug delivery and that spray drying can provide an efficient way to produce MPs to encapsulate cGAMP and stimulate the immune system.

In vivo evaluation of new adjuvant systems based on combination of Salmonella Typhi porins with particulate systems: Liposomes versus polymeric particles.

Subunit vaccines that have weak immunogenic activity require adjuvant systems for enhancedcellular and long-acting humoral immune responses. Both lipid-based and polymeric-based particulate adjuvants have been widely investigated to induce the desired immune responses against the subunit vaccines. The adjuvant efficacy of these particulate adjuvants depends upon their physicochemical properties such as particle size, surface charge, shape and their composition. Previously, we showed in vitro effect of adjuvant systems based on combination of chitosan and Salmonella Typhi porins in microparticle or nanoparticle form, which were spherical with positive surface charge. In the present study, we have further developed an adjuvant system based on combination of porins with liposomes (cationic and neutral) and investigated the adjuvant effect of both the liposomal and polymeric systems in BALB/c mice using a model antigen, ovalbumin. Humoral immune responses were determined following priming and booster dose at 15-day intervals. In overall, IgM and IgG levels were induced in the presence of both the liposomal and polymeric adjuvant systems indicating the positive impact of combination with porins. The highest IgM levels were obtained on Day 8, and liposomal adjuvant systems were found to elicit significantly higher IgM levels compared to polymeric systems. IgG levels were increased significantly after booster, particularly more profound with the micro-sized polymeric system when compared to cationic liposomal system with nano-size. Our results demonstrated that the developed particulate systems are promising both as an adjuvant and delivery system, providing enhanced immune responses against subunit antigens, and have the potential for long-term protection.

Development of a broadly active influenza intranasal vaccine adjuvanted with self-assembled particles composed of mastoparan-7 and CpG.

Currently licensed vaccine adjuvants offer limited mucosal immunity, which is needed to better combat respiratory infections such as influenza. Mast cells (MCs) are emerging as a target for a new class of mucosal vaccine adjuvants. Here, we developed and characterized a nanoparticulate adjuvant composed of an MC activator [mastoparan-7 (M7)] and a TLR ligand (CpG). This novel nanoparticle (NP) adjuvant was co-formulated with a computationally optimized broadly reactive antigen (COBRA) for hemagglutinin (HA), which is broadly reactive against influenza strains. M7 was combined at different ratios with CpG and tested for in vitro immune responses and cytotoxicity. We observed significantly higher cytokine production in dendritic cells and MCs with the lowest cytotoxicity at a charge-neutralizing ratio of nitrogen/phosphate = 1 for M7 and CpG. This combination formed spherical NPs approximately 200 nm in diameter with self-assembling capacity. Mice were vaccinated intranasally with COBRA HA and M7-CpG NPs in a prime-boost-boost schedule. Vaccinated mice had significantly higher antigen-specific antibody responses (IgG and IgA) in serum and mucosa compared with controls. Splenocytes from vaccinated mice had significantly increased cytokine production upon antigen recall and the presence of central and effector memory T cells in draining lymph nodes. Finally, co-immunization with NPs and COBRA HA induced influenza H3N2-specific HA inhibition antibody titers across multiple strains and partially protected mice from a challenge against an H3N2 virus. These results illustrate that the M7-CpG NP adjuvant combination can induce a protective immune response with a broadly reactive influenza antigen via mucosal vaccination.

Vinyl Sulfone-functionalized Acetalated Dextran Microparticles as a Subunit Broadly Acting Influenza Vaccine.

Influenza is a global health concern with millions of infections occurring yearly. Seasonal flu vaccines are one way to combat this virus; however, they are poorly protective against influenza as the virus is constantly mutating, particularly at the immunodominant hemagglutinin (HA) head group. A more broadly acting approach involves Computationally Optimized Broadly Reactive Antigen (COBRA). COBRA HA generates a broad immune response that is capable of protecting against mutating strains. Unfortunately, protein-based vaccines are often weekly immunogenic, so to help boost the immune response, we employed the use of acetalated dextran (Ace-DEX) microparticles (MPs) two ways: one to conjugate COBRA HA to the surface and a second to encapsulate cGAMP. To conjugate the COBRA HA to the surface of the Ace-DEX MPs, a poly(L-lactide)-polyethylene glycol co-polymer with a vinyl sulfone terminal group (PLLA-PEG-VS) was used. MPs encapsulating the STING agonist cGAMP were co-delivered with the antigen to form a broadly active influenza vaccine. This vaccine approach was evaluated in vivo with a prime-boost-boost vaccination schedule and illustrated generation of a humoral and cellular response that could protect against a lethal challenge of A/California/07/2009 in BALB/c mice.

CD4+ and CD8+ Circulating Memory T Cells Are Crucial in the Protection Induced by Vaccination with Salmonella Typhi Porins.

Salmonella enterica serovar Typhi (S. Typhi) porins, OmpC and OmpF, are potent inducers of the immune response against S. Typhi in mice and humans. Vaccination with porins induces the protection against 500 LD50 of S. Typhi, life-lasting bactericidal antibodies and effector T cell responses in mice; however, the nature of the memory T cell compartment and its contribution to protection remains unknown. In this work, we firstly observed that vaccination with porins induces in situ (skin) CD4+ and CD8+ T cell responses. Analysis of the porin-specific functional responses of skin CD4+ and CD8+ T cells showed IFN-gamma- and IL-17-producing cells in both T cell populations. The memory phenotype of porin-specific T cells indicated the presence of resident and effector memory phenotypes in the skin, and a central memory phenotype in the skin-draining lymph node. In addition, we demonstrated that vaccination with porins via skin reduces the bacterial burden following challenge. Finally, evaluating the role of the circulating T cell memory population in protection, we showed that circulating memory CD4+ and CD8+ T cells are crucial in porin-mediated protection against S. Typhi. Overall, this study highlights the importance of inducing circulating memory T cell responses in order to achieve the optimal protection provided by porins, showing a mechanism that could be sought in the rational development of vaccines.

Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan.

In order to improve the immunogenicity of the highly purified vaccine antigens, addition of an adjuvant to formulation, without affecting the safety of the vaccine, has been the key aim of the vaccine formulators. In recent years, adjuvants which are composed of a delivery system and immunopotentiators have been preferred to induce potent immune responses. In this study, we have combined Salmonella Typhi porins and chitosan to develop a new adjuvant system to enhance the immunogenicity of the highly purified antigens. Cationic gels, microparticle (1.69 ± 0.01 µm) and nanoparticles (337.7 ± 1.7 nm) based on chitosan were prepared with high loading efficiency of porins. Cellular uptake was examined by confocal laser scanning microscopy, and the macrophage activation was investigated by measuring the surface marker as well as the cytokine release in vitro in J774A.1 macrophage murine cells. Porins alone were not taken up by the macrophage cells whereas in combination with chitosan a significant uptake was obtained. Porins-chitosan combination systems were found to induce CD80, CD86 and MHC-II expressions at different levels by different formulations depending on the particle size. Similarly, TNF-α and IL-6 levels were found to increase with porins-chitosan combination. Our results demonstrated that combination of porins with chitosan as a particulate system exerts enhanced adjuvant effect, suggesting a promising adjuvant system for subunit vaccines with combined immunostimulating activity.

Challenges in the development of vaccines against COVID-19. / Retos en el desarrollo de vacunas contra COVID-19.

In the face of the urgent need for a COVID-19 vaccine, currently more than 90 vaccine candidates are being developed using different strategies, such as inactivated or attenuated SARS-CoV-2 virus, viral vectors expressing antigens of this virus, nucleic acids or purified viral proteins. These vaccines are in preclinical development and at least six of them have already been injected into volunteers in safety clinical trials. However, the characteristics of the protective immune responses are still unknown; therefore, there is not evidence to indicate that these vaccines will induce protection and if this will be long-lasting. The development of SARS-CoV-2 vaccines is vital; nevertheless, it is also important to unveil the characteristics of the protective immune responses to guide the design of a vaccine that generates a long-lasting protection against COVID-19.

Ante la urgente necesidad de una vacuna contra el virus causante de COVID-19, actualmente se han desarrollado más de 90 vacunas candidatas a partir de diferentes estrategias, como el uso del virus SARS-CoV-2 inactivado o atenuado, vectores virales que expresan antígenos de este virus, ácidos nucleicos o proteínas virales purificadas. Estas vacunas se encuentran en estudios preclínicos y al menos seis de ellas ya han sido inyectadas en voluntarios de ensayos clínicos de seguridad. Sin embargo, aún se desconocen las características de la respuesta inmune protectora y, por lo tanto, no hay evidencia que indique si estas vacunas lograrán inducir inmunidad y si esta será de larga duración. El desarrollo de vacunas contra el SARS-CoV-2 es imperante; no obstante, lo es también determinar las características de la respuesta inmune protectora para que guíe el diseño de una vacuna que genere protección de larga duración contra el COVID-19.

The role of T Cells in SARS-CoV-2 Infection. / El papel de los linfocitos T en la infección por SARS-CoV-2.

T lymphocytes (or T cells) are characterized by having an essential role in the control of acute viral infections. SARS-CoV-2 infection is an acute viral infection that mainly affects the respiratory tract causing COVID-19 disease, which presents with mild, moderate and critical symptoms that can lead to the death of the patient. The induction of populations of CD4+ and CD8+ T cell with a functional memory phenotype could be decisive in the control of viral replication and therefore be determinants in the course of the disease. In this opinion article, we will review the reported evidence regarding the presence, phenotype, and function of circulating T cell populations and the site of infection to understand their possible role in controlling viral replication, in the severity of the disease, and the importance of T-cell-mediated protection in the development of vaccines against SARS-CoV-2 infection.

Los linfocitos T se caracterizan por tener un papel esencial en el control de infecciones virales agudas. La infección por SARS-CoV-2 es una infección viral aguda que afecta principalmente el tracto respiratorio y causa la enfermedad COVID-19, la cual cursa con síntomas leves, moderados y críticos que pueden llevar a la muerte del paciente. La inducción de poblaciones de linfocitos T CD4+ y CD8+ con fenotipos de memoria funcionales podrían ser esenciales en el control de la replicación viral y, por lo tanto, determinantes en el curso de la enfermedad. En este artículo de opinión revisaremos las evidencias reportadas en cuanto a la presencia, fenotipo y función de las poblaciones de linfocitos T en circulación y en el sitio de infección para entender su posible papel en el control de la replicación viral, en la severidad de la enfermedad y la importancia de la protección mediada por linfocitos T en el desarrollo de vacunas contra la infección por SARS-CoV-2.