To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.

Porcine epidemic diarrhea virus (PEDV) causes acute watery diarrhea and high mortality in newborn piglets. Activation of intestinal mucosal immunity is crucial to anti-PEDV infection. To develop a vaccine capable of stimulating intestinal mucosal immunity, we prepared a bacterium (Lactococcus lactis)-like particle (BLP) vaccine (S1-BLPs) displaying the S1 protein, a domain of PEDV spike protein (S), based on gram-positive enhancer matrix (GEM) particle display technology. We further compared the effects of different vaccination routes on mucosal immune responses in mice induced by S1-BLPs. The specific IgG titer in serum of intramuscularly immunized mice with S1-BLPs was significantly higher than that of the intranasally administered. The specific IgA antibody was found in the serum and intestinal lavage fluid of mice vaccinated intranasally, but not intramuscularly. Moreover, the intranasally inoculated S1-BLPs induced higher levels of IFN-γ and IL-4 in serum than the intramuscularly inoculated. In addition, the ratio of serum IgG2a/IgG1 of mice inoculated intramuscularly was significantly higher with S1-BLPs compared to that of with S1 protein, suggesting that the immune responses induced by S1-BLPs was characterized by helper T (Th) cell type 1 immunity. The results indicated that S1-BLPs induced systemic and local immunity, and the immunization routes significantly affected the specific antibody classes and Th immune response types. The intranasally administered S1-BLPs could effectively stimulate intestinal mucosal specific secretory IgA response. S1-BLPs have the potential to be developed as PEDV mucosal vaccine.

Vaccines are the most effective tool to control infectious diseases, which provoke significant morbidity and mortality rates. Most vaccines are administered through the parenteral route and can elicit a robust systemic humoral response, but they induce a weak T-cell-mediated immunity and are poor inducers of mucosal protection. Considering that most pathogens enter the body through mucosal surfaces, a vaccine that elicits protection in the first site of contact between the host and the pathogen is promising. However, despite the advantages of mucosal vaccines as good options to confer protection on the mucosal surface, only a few mucosal vaccines are currently approved. In this review, we discuss the impact of vaccine administration in different mucosal surfaces; how appropriate adjuvants enhance the induction of protective mucosal immunity and other factors that can influence the mucosal immune response to vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as the prime challenge facing public health safety since 2019. Correspondingly, coronavirus disease 2019 (COVID-19) vaccines have been developed and administered worldwide, varying in design strategies, delivery routes, immunogenicity and protective efficacy. Here, a replication-competent vesicular stomatitis virus (VSV) vectored recombinant COVID-19 vaccine was constructed and evaluated in BALB/c mice and Syrian golden hamsters. In BALB/c mice, intramuscular (i.m.) inoculation of recombinant vaccine induced significantly higher humoral immune response than that of the intranasal (i.n.) inoculation group. Analyses of cellular immunity revealed that a Th1-biased cellular immune response was induced in i.n. inoculation group while both Th1 and Th2 T cells were activated in i.m. inoculation group. In golden hamsters, i.n. inoculation of the recombinant vaccine triggered robust humoral immune response and conferred prominent protective efficacy post-SARS-CoV-2 challenge, indicating a better protective immunity in the i.n. inoculation group than that of the i.m. inoculation group. This study provides an effective i.n.-delivered recombinant COVID-19 vaccine candidate and elucidates a route-dependent manner of this vaccine candidate in two most frequently applied small animal models. Moreover, the golden hamster is presented as an economical and convenient small animal model that precisely reflects the immune response and protective efficacy induced by replication-competent COVID-19 vaccine candidates in other SARS-CoV-2 susceptible animals and human beings, especially in the exploration of i.n. immunization.

Vaccines containing multiple antigens may induce broader immune responses and provide better protection against Mycobacterium tuberculosis (Mtb) infection as compared to a single antigen. However, strategies for incorporating multiple antigens into a single vector and the immunization routes may affect their immunogenicity. In this study, we utilized recombinant adenovirus type 5 (rAd5) as a model vaccine vector, and Ag85A (Rv3804c) and Mtb32 (Rv0125) as model antigens, to comparatively evaluate the influence of codon usage optimization, signal sequence, fusion linkers, and immunization routes on the immunogenicity of tuberculosis (TB) vaccine containing multiple antigens in C57BL/6 mice. We showed that codon-optimized Ag85A and Mtb32 fused with a GSG linker induced the strongest systemic and pulmonary cell-mediated immune (CMI) responses. Strong CMI responses were characterized by the generation of a robust IFN-γ ELISPOT response as well as antigen-specific CD4(+) T and CD8(+) T cells, which secreted mono-, dual-, or multiple cytokines. We also found that subcutaneous (SC) and intranasal (IN)/oral immunization with this candidate vaccine exhibited the strongest boosting effects for Mycobacterium bovis bacille Calmette-Guérin (BCG)-primed systemic and pulmonary CMI responses, respectively. Our results supported that codon optimized Ag85A and Mtb32 fused with a proper linker and immunized through SC and IN/oral routes can generate the strongest systemic and pulmonary CMI responses in BCG-primed mice, which may be particularly important for the design of TB vaccines containing multiple antigens.

The mucosal immune system displays several adaptations reflecting the exposure to the external environment. The efficient induction of mucosal immune responses also requires specific approaches, such as the use of appropriate administration routes and specific adjuvants and/or delivery systems. In contrast to vaccines delivered via parenteral routes, experimental, and clinical evidences demonstrated that mucosal vaccines can efficiently induce local immune responses to pathogens or tumors located at mucosal sites as well as systemic response. At least in part, such features can be explained by the compartmentalization of mucosal B and T cell populations that play important roles in the modulation of local immune responses. In the present review, we discuss molecular and cellular features of the mucosal immune system as well as novel immunization approaches that may lead to the development of innovative and efficient vaccines targeting pathogens and tumors at different mucosal sites.