Outer Membrane Vesicles: A Challenging Yet Promising Platform for COVID-19 Vaccines

The outer membrane vesicles (OMVs) are vesicles released from Gram-negative bacteria, which present a range of biological applications, such as vaccine adjuvants. OMVs present several pathogen-associated molecular patterns, being immunogenic and capable of triggering different arms of the immune response. Thus, they are suitable for mucosal and parenteral delivery, feasible to obtain and have been used in licensed-vaccines previously. However, the extraction protocols and manipulations can modify OMVs cargo and, consequentially, the immunization results. Therefore, this chapter will review OMVs use as adjuvant and discuss results from COVID-19 vaccines which employed this technique

Trained-immunity and cross-reactivity for protection: insights from the coronavirus disease 2019 and monkeypox emergencies for vaccine development

The emergence and re-emergence of pathogens is a public-health concern, which has become more evident after the coronavirus disease 2019 (COVID-19) pandemic and the monkeypox outbreaks in early 2022. Given that vaccines are the more effective and affordable tools to control infectious diseases, the authors reviewed two heterologous effects of vaccines: the trained immunity and the cross-reactivity. Trained immunity, provided by attenuated vaccines, was exemplified in this article by the decreased the burden of COVID-19 in populations with high Bacille Calmette-Guerin (BCG) coverage. Cross-reactive responses were exemplified here by the studies which suggested that vaccinia could help controlling the monkeypox outbreak, because of common epitopes shared by orthopoxviruses. Although modern vaccination is likely to use subunit vaccines, the authors discussed how adjuvants might be the key to induce trained immunity and improve cross-reactive responses, ensuring that heterologous effects would improve the vaccine’s response

Parenteral and mucosal immunization with Outer Membrane Vesicles from Neisseria meningitidis serogroup C aiming cross-reactivity with serogroup W in mice.

A doença meningocócica invasiva (DMI), provocada por Neisseria meningitidis, ocorre globalmente, contudo, regiões em desenvolvimento sofrem os maiores impactos da doença. No Brasil, os principais sorogrupos isolados são B e C, e observa-se a emergência do sorogrupo W. Esse trabalho propõe o estudo, em camundongos, da imunização com vesículas de membrana externa (OMVs) de cepa brasileira de N. meningitidis C que apresenta as proteínas porinas 2a e P1.5, comuns ao sorogrupo W; sendo as cepas utilizadas para o estudo classificadas como C:2a:P1.5 e W:2a:P1.5,2. Fêmeas adultas, da linhagem A/Sn (H2a), foram imunizadas por sistema prime-booster utilizando 4 doses intranasais (IN) e reforço intramuscular (IM) com OMVs associadas à toxina colérica subunidade B (TCB) ou por sistema de duas doses IM com OMVs associadas ao hidróxido de alumínio (HA). Os animais foram acompanhados até que fossem idosos. A resposta imune foi avaliada por ELISA, Immunoblotting, Dot-blot, Imunohistoquímica e ELISpot. Os adjuvantes contribuíram para indução de maiores títulos de anticorpos. OMV+TCB induziu títulos de anticorpos estatisticamente superiores ao controle pré-imune após as 4 doses IN e manteve superioridade em todas as faixas etárias. OMV+HA alcançou diferença estatística em relação ao controle após a dose de reforço e manteve a diferença até a meia-idade. Todos os grupos apresentaram IgG de índice de avidez elevado, independente do uso de adjuvantes. A razão IgG1/IgG2a foi intermediária em grupos imunizados com OMVs sozinhas e alta em grupos imunizados com OMVs associadas a adjuvantes. Os anticorpos reconheceram antígenos de pesos moleculares atribuídos à Porina A, importante para proteção específica contra a cepa de imunização, à Adesina A de Neisseria e à Proteína de ligação à transferrina, importantes para reatividade cruzada com cepas diferentes. Os anticorpos reconheceram antígenos da cepa heteróloga W:2a:P1.5,2, além de cepas representativas da circulação dos sorogrupos B, C e Y no Brasil. A caracterização celular dos baços, feita pelos marcadores anti-CD68, anti-CD4, anti-CD79 e anti-CD25, não apontou diferenças entre os camundongos idosos e indivíduo adulto. Também não se observou diferenças na população celular de grupos imunes e controles, porém, esplenócitos dos camundongos imunes, idosos, produziram IL-4 e IL-17 quando estimulados com as OMVs e com o polissacarídeo capsular C de meningococo. Por fim, concluiu-se que o uso de adjuvantes e a administração de doses de reforço favoreceram a elevação dos títulos de anticorpos e sua manutenção por períodos maiores; estimulou a diferenciação da resposta imune em perfil Th2; levou ao reconhecimento de mais antígenos e à secreção de maiores quantidades de citocinas.Invasive meningococcal disease (IMD), caused by Neisseria meningitidis, occurs globally, however, developing countries deal with greater burden of the disease. In Brazil, the main serogroup isolated is serogroup C, and the emergence of serogroup W has been seen. This work studied, in mice, the immunization with outer membrane vesicles (OMVs) of a Brazilian strain of N. meningitidis C that presents porin proteins 2a and P1.5, common to serogroup W; the phenotypic characterization of the strains used was C:2:a:P1.5 and W:2a:P1.5,2. Adult females of A/Sn (H2a) mice were immunized by prime-booster regimen with 4 intranasal (IN) doses and one intramuscular (IM) booster dose with OMVs associated with cholera toxin subunit B (CTB); or by two IM doses regimen with OMVs associated with aluminium hydroxide (AH). The mice were followed until old age. The immune response was evaluated by ELISA, Immunoblotting, Dot-blot, Immunohistochemistry and ELISpot. The adjuvants contributed to the induction of higher antibody titers. OMV+CTB triggered antibodies titers statistically higher than pre-immune control following 4 IN doses and maintained this superiority during all ages. OMV+AH needed a booster dose to reach statistical difference in comparison with control, maintaining it until middle-age. All groups presented a high avidity index of IgG, regardless of the adjuvant use. The IgG1/IgG2a ratio was intermediary in groups immunized with OMVs alone and higher in groups immunized with OMVs associated with adjuvants. The antibodies recognized antigens of molecular weight attributed to porin A, an important protein to respond specifically against the immunization strain, to Neisserial Adhesin A and Transferrin-binding proteins, important for cross-reactivity with different strains. The antibodies also recognized antigens of heterologous strain W:2a:P1.5,2 and strains which represent the circulation of serogroups B, C and Y in Brazil. The cellular characterization of spleens, marked with anti-CD68, anti-CD4, anti-CD79 and anti-CD25, did not point to differences between old and adult mice. Also, it was not observed difference between the cellular populations of immune and control groups, however, splenocytes of immunized, elderly mice, produced IL-4 and IL-17 when stimulated with OMVs and capsular polysaccharide C of meningococci. It was concluded that adjuvant use and booster doses improved the antibody titers and their persistence for longer periods, provided a Th2 type of immune response, led to the recognition of more antigens and the secretion of higher quantities of cytokines

Enzyme-Linked Immunosorbent Assay: An Adaptable Methodology to Study SARS-CoV-2 Humoral and Cellular Immune Responses

The Enzyme-Linked Immunosorbent Assay is a versatile technique, which can be used for several applications. It has enormously contributed to the study of infectious diseases. This review highlights how this methodology supported the science conducted in COVID-19 pandemics, allowing scientists to better understand the immune response against SARS-CoV-2. ELISA can be modified to assess the functionality of antibodies, as avidity and neutralization, respectively by the standardization of avidity-ELISA and surrogate-neutralization methods. Cellular immunity can also be studied using this assay. Products secreted by cells, like proteins and cytokines, can be studied by ELISA or its derivative Enzyme-linked immunospot (ELISpot) assay. ELISA and ELISA-based methods aided the area of immunology against infectious diseases and is still relevant, for example, as a promising approach to study the differences between natural and vaccine-induced immune responses against SARS-CoV-2

Animal Models to Test SARS-CoV-2 Vaccines: Which Ones Are in Use and Future Expectations

Since late 2019 and early 2020, with the emergence of the COVID-19 pandemic, scientists are rushing to develop treatment and prevention methods to combat SARS-CoV-2. Among these are vaccines. In view of this, the use of animals as experimental models, both to investigate the immunopathology of the disease and to evaluate the efficacy and safety of vaccines, is mandatory. This work aims to describe, through recent scientific articles found in reliable databases, the animal models used for the in vivo testing of COVID-19 vaccines, demonstrating some possibilities of more advantageous/gold-standard models for SARS-CoV-2 vaccines. The majority of the studies use rodents and primates. Meanwhile, the most adequate model to be used as the gold standard for in vivo tests of COVID-19 vaccines is not yet conclusive. Promising options are being discussed as new tests are being carried out and new SARS-CoV-2 variants are emerging