Th1 and Th17 mucosal immune responses elicited by nasally inoculation in mice with virulence factors of Mycoplasma hyopneumoniae.

Nicotinamide Adenine Dinucleotide-Dependent (NADH) flavin oxidoreductase and NADH oxidase (NOX) are important virulence factors of Mycoplasma hyopneumoniae (Mhp), which are devoted to the function of adhesion, oxidative stress damage and apoptosis to host cells in our previous studies. Here, immune responses of NADH flavin oxidoreductase (NFOR) and NOX in mice and immune efficacy inoculated with intramuscular (IM), intranasal (IN), intramuscular unite intranasal (IM + IN) approaches were evaluated and compared. Cellular immunity levels, systemic immune and local mucosal immune responses were investigated by indirect enzyme-linked immunosorbent assay (iELISA) and quantitative reverse transcription PCR (qRT-PCR). Mice inoculated with NFOR and NOX by IM and IN or IM + IN could induce obvious secretion of specific immunoglobulin G (IgG) and secretory immunoglobulin A antibodies (sIgA) compared to those in negative control group. IM + IN inoculation resulted in systemic and local mucosal immune responses that were strongly produced. Moreover, Mhp NFOR and NOX could activate local mucosal immune responses mediated by Th1 and Th17 cells by IN. Our finding supported the notion that IM + IN was an effective immunization route for Mhp, which lays a foundation for more effective prevention of Mhp, and provides theoretical basis for the development of new subunit vaccines of Mhp.

Comparison of the efficacy and safety of different immunization routes induced by human respiratory syncytial virus F protein with CpG adjuvant in mice.

Human respiratory syncytial virus (HRSV) is a leading cause worldwide of severe respiratory illness in infants and the elderly. The ideal HRSV vaccine should induce a systemic immune response, especially mucosal immunity. In this study, mice were immunized twice with F protein combined with CpG adjuvant to compare the safety and efficacy of 4 immunization routes, including intranasal primed/intramuscular boosted immunization (CpG + F/in+im), intramuscular primed/intranasal boosted immunization (CpG + F/im+in), intramuscular primed/intramuscular boosted immunization (CpG + F/im + im) and intranasal primed/intranasal boosted immunization (CpG + F/in+in). Compared with the control group (CpG/in+im, CpG/im+in, CpG/im + im and CpG/in+in), all 4 immunization routes induced a high titer of neutralizing antibodies and a strong cellular immune response. Mice in the CpG + F/in+in group induced the highest antibody neutralization titer, and IgA antibody in bronchoalveolar lavage fluid (BALF) was the highest. The copy of HRSVs in the lung decreased by approximately 3 log10. As seen from the IgG1/IgG2a and IFN-γ/IL-4-secreting lymphocyte ratios, compared with the mice in the CpG + F/im + im group, mice in the CpG + F/in+in group induced Th1-baised humoral and cellular immune responses and significantly reduced lung pathological injury. In conclusion, among the 4 immunization routes, the safety and efficacy induced by the mice in the CpG + F/in+in group were the best. We can conclude that intranasal immunization is superior to intramuscular immunization using F protein with CpG adjuvant as vaccine candidates.

Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

MERS-CoV Spike Protein Vaccine and Inactivated Influenza Vaccine Formulated with Single Strand RNA Adjuvant Induce T-Cell Activation through Intranasal Immunization in Mice.

The effectiveness of vaccines is enhanced by adding adjuvants. Furthermore, the selection of an inoculation route depends on the type of adjuvant used and is important for achieving optimum vaccine efficacy. We investigated the immunological differences between two types of vaccines-spike protein from the Middle East respiratory syndrome virus and inactivated influenza virus vaccine, in combination with a single-stranded RNA adjuvant-administered through various routes (intramuscular, intradermal, and intranasal) to BALB/c mice. Intramuscular immunization with the RNA adjuvant-formulated spike protein elicited the highest humoral immune response, characterized by IgG1 and neutralizing antibody production. Although intranasal immunization did not elicit a humoral response, it showed extensive T-cell activation through large-scale induction of interferon-γ- and interleukin-2-secreting cells, as well as CD4+ T-cell activation in mouse splenocytes. Moreover, only intranasal immunization induced IgA production. When immunized with the inactivated influenza vaccine, administration of the RNA adjuvant via all routes led to protection after viral challenge, regardless of the presence of a vaccine-specific antibody. Therefore, the inoculation route should depend on the type of immune response needed; i.e., the intramuscular route is suitable for eliciting a humoral immune response, whereas the intranasal route is useful for T-cell activation and IgA induction.

Skeletal Muscle Is an Antigen Reservoir in Integrase-Defective Lentiviral Vector-Induced Long-Term Immunity.

We previously developed integrase-defective lentiviral vectors (IDLVs) as an antigen delivery system for inducing strong and prolonged immunity in animal models. Here, we examined the association between persistence of antigen expression and durability of immune response. Following a single intramuscular (i.m.) or subcutaneous (s.c.) injection of IDLV delivering GFP in mice, we evaluated antigen expression and inflammation at the site of injection and persistence of antigen-specific T cells at early and late time points. Durable antigen expression was detected up to 90 days only after i.m. immunization. Mononuclear inflammation was evident soon after IDLV injection in both i.m. and s.c. immunized mice, but remained detectable up to 30 days postinjection only in i.m. immunized mice. Similarly, GFP-specific T cells were more persistent in the i.m. immunized mice. Interestingly, GFP+ muscle fibers were co-expressing major histocompatibility complex (MHC) class I, suggesting that muscle cells are competent for presenting antigens to T cells in vivo. In in vitro experiments, we demonstrated that although both primary myoblasts and myocytes present the antigen to GFP-specific T cells through MHC class I, myoblasts are more resistant to Fas-dependent cytotoxic T lymphocyte (CTL) killing activity. Overall, these data indicate that muscle cells may serve as an antigen reservoir that contributes to the long-term immunity induced by IDLV vaccination.

Soluble Recombinant Hemagglutinin Protein of H1N1pdm09 Influenza Virus Elicits Cross-Protection Against a Lethal H5N1 Challenge in Mice.

Currently, influenza vaccines are produced using embryonated chicken eggs. Recently, recombinant influenza vaccines have been developed as a potential alternative to egg-grown vaccines. In this study, we evaluated the efficacy of soluble recombinant hemagglutinin (HA) protein produced in human cell culture (Expi293F cells) as an influenza vaccine against homosubtypic and heterosubtypic influenza virus challenges in mice. Mice were immunized intramuscularly with purified soluble HA protein of H1N1pdm09 virus and then challenged with a lethal dose of H1N1pdm09, seasonal H3N2, or highly pathogenic avian influenza (HPAI) H5N1 virus. Vaccinated mice showed better morbidity than mock-vaccinated mice following H1N1pdm09 challenge. By contrast, all mice died following H3N2 challenge. Interestingly, all vaccinated mice survived challenge with H5N1 virus, whereas all mock-vaccinated mice died. These results suggest that intramuscular immunization with recombinant HA proteins produced in Expi 293F cells could be of value in influenza vaccine strategies.

Immunomodulatory activity of wild Artemisia rupestris L. crude polysaccharide as an adjuvant / 国际生物医学工程杂志

Objective@#To investigate the enhancement effect of Xinjiang wild Artemisia rupestris L. crude polysaccharides (WARCP) as an adjuvant on ovalbumin (OVA) vaccine in mice immunized intramuscularly.@*Methods@#ICR mice were randomly divided into 6 groups (5 per group), including 9 g/L NaCl group (blank control), OVA group (10 μg OVA), low dose WARCP/OVA group (OVA+50 μg WARCP), medium dose WARCP/OVA group (OVA+200 μg WARCP), high dose WARCP/OVA group (OVA+400 μg WARCP), and aluminum adjuvant (Alum)/OVA group (positive control group, OVA+100 μg Alum). ICR mice were immunized intramuscularly and weighted. The OVA-specific antibodies and subtypes in serum were detected by enzyme linked immunosorbent assay (ELISA). T cells subsets from spleen and lymph nodes were detected by flow cytometry.@*Results@#The medium-dose WARCP/OVA group enhanced IgG and IgG1 levels and increased early antibody levels (all P<0.05). The medium-dose WARCP/OVA group and the high-dose WARCP/OVA group significantly enhanced IgG2a levels (all P<0.05), but the difference was not statistically significant comparing with Alum/OVA group (P>0.05). The low-dose WARCP/OVA group enhanced the percentage of CD4+ T cells in spleen and CD4+ T, CD8+ T, CD4+CD44+ T cells in lymph nodes (all P<0.05). The medium dose WARCP/OVA group and the high dose WARCP/OVA group enhanced the CD4+ T, CD8+ T, CD4+CD44+ T, CD8+CD44+ T cells in spleen and CD8+CD44+ T cell in lymph nodes (all P<0.05).@*Conclusions@#Plant-derived WARCP as an OVA protein vaccine adjuvant can enhance cellular immunity and humoral immunity, and it is safe and reliable. The results in this study provide a theoretical basis for the popularization and application of WARCP.

Immunomodulatory activity of wild Artemisia rupestris L. crude polysaccharide as an adjuvant / 国际生物医学工程杂志

Objective To investigate the enhancement effect of Xinjiang wild Artemisia rupestris L. crude polysaccharides (WARCP) as an adjuvant on ovalbumin (OVA) vaccine in mice immunized intramuscularly. Methods ICR mice were randomly divided into 6 groups (5 per group), including 9 g/L NaCl group (blank control), OVA group (10 μg OVA), low dose WARCP/OVA group (OVA+50 μg WARCP), medium dose WARCP/OVA group (OVA+200 μg WARCP), high dose WARCP/OVA group (OVA+400 μg WARCP), and aluminum adjuvant (Alum)/OVA group (positive control group, OVA +100 μg Alum). ICR mice were immunized intramuscularly and weighted. The OVA-specific antibodies and subtypes in serum were detected by enzyme linked immunosorbent assay (ELISA). T cells subsets from spleen and lymph nodes were detected by flow cytometry. Results The medium-dose WARCP/OVA group enhanced IgG and IgG1 levels and increased early antibody levels (all P<0.05). The medium-dose WARCP/OVA group and the high-dose WARCP/OVA group significantly enhanced IgG2a levels (all P<0.05), but the difference was not statistically significant comparing with Alum/OVA group (P>0.05). The low-dose WARCP/OVA group enhanced the percentage of CD4+ T cells in spleen and CD4 + T, CD8+ T, CD4 +CD44 + T cells in lymph nodes (all P<0.05). The medium dose WARCP/OVA group and the high dose WARCP/OVA group enhanced the CD4 + T, CD8 + T, CD4 +CD44 + T, CD8 +CD44+ T cells in spleen and CD8+CD44+ T cell in lymph nodes (all P<0.05). Conclusions Plant-derived WARCP as an OVA protein vaccine adjuvant can enhance cellular immunity and humoral immunity, and it is safe and reliable. The results in this study provide a theoretical basis for the popularization and application of WARCP.

Evaluation of N-phosphonium chitosan as a novel vaccine carrier for intramuscular immunization.

Chitosan, as a potential vaccine delivery material, has obtained much attention for immunization prevention and therapy. However, its poor water solubility brings inconvenience for the practical applications. To address this issue, researchers have carried out many chemical modifications to prepare water-soluble chitosan derivatives for vaccine delivery. In this work, we prepared a chitosan derivative N-phosphonium chitosan with excellent water solubility and explored its potential as an intramuscular vaccine delivery system by using ovalbumin as a model antigen. Different vaccine formulations were intramuscularly injected into test mice. Through an immunohistochemistry assay, N-phosphonium chitosan-based antigen formulation could promote antigen arrival from injection site to the secondary lymph organ spleen. Further immunization results showed that 1 mg/ml N-phosphonium chitosan-based vaccine formulation could contribute to significantly higher level of antigen-specific immune responses, including higher antigen-specific IgG antibody titer, splenocyte proliferation, and cytokines secretion (interferon-γ, interleukin-10, and interleukin-4) by the splenocytes of the immunized mice. From the results, the water-soluble chitosan derivative N-phosphonium chitosan could be developed as a potential antigen carrier for immunization prevention and therapy.

Thermo-sensitive hydrogel PLGA-PEG-PLGA as a vaccine delivery system for intramuscular immunization.

In this work, we explored the potential of thermo-sensitive PLGA-PEG-PLGA with sol-gel transition temperature around 32℃ as an intramuscular vaccine delivery system by using ovalbumin as a model antigen. First, in vitro release test showed that the PLGA-PEG-PLGA-deriving hydrogels could release ovalbumin in vitro in a more sustainable way. From fluorescence living imaging, 50-200 mg/mL of PLGA-PEG-PLGA formulations could release antigen in a sustainable manner in vivo, suggesting that the PLGA-PEG-PLGA hydrogel worked as an antigen-depot. Further, the sustainable antigen release from the PLGA-PEG-PLGA hydrogels increased antigen availability in the spleens of the immunized mice. The intramuscular immunization results showed that 50-200 mg/mL of PLGA-PEG-PLGA formulations promoted significantly more potent antigen-specific IgG immune response. In addition, 200 mg/mL of PLGA-PEG-PLGA formulation significantly enhanced the secretion of both Th1 and Th2 cytokines. From in vitro splenocyte proliferation assay, 50-200 mg/mL of PLGA-PEG-PLGA formulations all initiated significantly higher splenocyte activation. These results indicate that the thermo-sensitive and injectable PLGA-PEG-PLGA hydrogels (particularly, 200 mg/mL of PLGA-PEG-PLGA-based hydrogel) own promising potential as an intramuscular vaccine delivery system.

Influences of intramuscular immunization with cultivated Cistanche deserticola crude polysaccharides on efficacy of ovalbumin(OVA) protein / 中华微生物学和免疫学杂志

Objective To investigate the immunopotentiating effects of cultivated Cistanche deser-ticola (C.deserticola) crude polysaccharides (CCDCP) as an adjuvant on the model antigen ovalbumin (OVA). Methods Low,medium and high doses of CCDCP in combination with OVA were intramuscularly injected twice into ICR mice at an interval of two weeks,respectively. Aluminum adjuvant was used to set up positive control group. Levels of IgG,IgG1 and IgG2a antibodies were detected by ELISA. Splenocyte prolif-eration was detected by MTT assay. Growth conditions of the immunized mice were observed. Results IgG level was significantly increased in the high dose group 7 days after the first immunization(P<0.05),espe-cially on the 21st and 28th days (P<0.01) as compared with that of the aluminum adjuvant group. High dose of CCDCP in combination with OVA significantly up-regulated the levels of IgG1 and IgG2a in mice as compared with immunization with OVA alone (P<0.05). Moreover, IgG2a level in mice immunized with high dose of CCDCP and OVA was higher than that of the aluminum adjuvant group(P<0.05). Splenocyte proliferation was significantly enhanced in the medium and low dose groups in comparison with that of the OVA group (P<0.05) and the aluminum adjuvant group (P<0.01). No significant difference in mouse body weight was observed in different groups(P>0.05). Conclusion CCDCP as an adjuvant of OVA pro-tein vaccine can enhance Th1 and Th2 immune responses,especially the early antibody production and Th1 immune response. These results will provide some information for further studies of CCDCP as a vaccine ad-juvant.

Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) vaccine strain 168 is an intrapulmonically injected attenuated live vaccine that is available in the Chinese market. The aim of this study was to develop suitable adjuvants for this live vaccine to provide effective protection after intramuscular inoculation. Several adjuvant components were screened to assess their toxicity for the live vaccine, and various adjuvant formulations were then designed and prepared. Vaccines supplemented with these adjuvants were used to immunize mice intramuscularly to assess the capacity of the adjuvants to induce a specific immune response. The screened formulations were then evaluated in pigs. Seven of the eight adjuvant components did not affect the viability of the live vaccine, and seven different adjuvant formulations were then designed. In mice, the ISCOM-matrix adjuvant and the levamisole-chitosan mixture adjuvant significantly enhanced serum IgG responses against M. hyopneumoniae, while lymphocyte proliferation was enhanced by the ISCOM-matrix adjuvant, the carbomer-astragalus polysaccharide mixture adjuvant and an oil-in-water emulsion adjuvant. These four adjuvants were evaluated in pigs. Enhancement of specific lymphocyte proliferation responses was observed in the groups vaccinated with the ISCOM-matrix adjuvant and the carbomer-astragalus polysaccharide mixture adjuvant. Significant enhancement of serum IgG antibody production was observed before challenge in pigs vaccinated with the carbomer-astragalus polysaccharide mixture adjuvant and the levamisole-chitosan mixture adjuvant, while after challenge, all of the animals that received vaccines containing adjuvants had higher antibody concentrations against M. hyopneumoniae than unvaccinated animals. Animals inoculated with a vaccine containing the ISCOM-matrix adjuvant (median score 3.57) or the carbomer-astragalus polysaccharide mixture adjuvant (median score 5.28) had reduced lesion scores compared to unvaccinated animals (median score 14.81). These studies will help in the development of appropriate adjuvants for intramuscular administration of this live M. hyopneumoniae vaccine.