Differential Immune Response Patterns Induced by Anionic and Cationic Lipid Adjuvants in Intranasal Anti-Influenza Immunization.

Currently, vaccine development against different respiratory diseases is at its peak. It is of utmost importance to find suitajble adjuvants that can increase the potency of the vaccine candidates. This study aimed to determine the systemic and splenic immune mechanisms in mice models induced by anionic and cationic lipid adjuvants in the presence of the vaccine-candidate influenza antigen hemagglutinin (HA). In the presence of the HA antigen, the cationic adjuvant (N3) increased conventional dendritic cell 1 (cDC1) abundance with enhanced MHCI and CD80-CD86 costimulatory marker expression, and significantly higher CD8T and Th17 populations with enhanced interferon-gamma (IFNγ) expression in CD8T and CD4T populations. Conversely, the anionic adjuvant (L3) increased the cDC2 population percentage with significantly higher MHCII and DEC205 expression, along with an increase in the CD4T and regulatory T cell populations. The L3-treated group also exhibited higher percentages of activated B and plasma cell populations with significantly higher antigen-specific IgG and IgA titer and virus neutralization potential. While the anionic adjuvant induced significantly higher humoral responses than the cationic adjuvant, the latter influenced a significantly higher Th1/Th17 response. For customized vaccine development, it is beneficial to have alternative adjuvants that can generate differential immune responses with the same vaccine candidate antigen. This study will aid the selection of adjuvants based on their charges to improve specific immune response arms in the future development of vaccine formulation.

Intranasal Coronavirus SARS-CoV-2 Immunization with Lipid Adjuvants Provides Systemic and Mucosal Immune Response against SARS-CoV-2 S1 Spike and Nucleocapsid Protein.

In this preclinical two-dose mucosal immunization study, using a combination of S1 spike and nucleocapsid proteins with cationic (N3)/or anionic (L3) lipids were investigated using an intranasal delivery route. The study showed that nasal administration of low amounts of antigens/adjuvants induced a primary and secondary immune response in systemic IgG, mIL-5, and IFN-gamma secreting T lymphocytes, as well as humoral IgA in nasal and intestinal mucosal compartments. It is believed that recipients will benefit from receiving a combination of viral antigens in promoting a border immune response against present and evolving contagious viruses. Lipid adjuvants demonstrated an enhanced response in the vaccine effect. This was seen in the significant immunogenicity effect when using the cationic lipid N3. Unlike L3, which showed a recognizable effect when administrated at a slightly higher concentration. Moreover, the findings of the study proved the efficiency of an intranasally mucosal immunization strategy, which can be less painful and more effective in enhancing the respiratory tract immunity against respiratory infectious diseases.

Long-Lasting Mucosal and Systemic Immunity against Influenza A Virus Is Significantly Prolonged and Protective by Nasal Whole Influenza Immunization with Mucosal Adjuvant N3 and DNA-Plasmid Expressing Flagellin in Aging In- and Outbred Mice.

Background: Vaccination is commonly used to prevent and control influenza infection in humans. However, improvements in the ease of delivery and strength of immunogenicity could markedly improve herd immunity. The aim of this pre-clinical study is to test the potential improvements to existing intranasal delivery of formalin-inactivated whole Influenza A vaccines (WIV) by formulation with a cationic lipid-based adjuvant (N3). Additionally, we combined WIV and N3 with a DNA-encoded TLR5 agonist secreted flagellin (pFliC(-gly)) as an adjuvant, as this adjuvant has previously been shown to improve the effectiveness of plasmid-encoded DNA antigens. Methods: Outbred and inbred mouse strains were intranasally immunized with unadjuvanted WIV A/H1N1/SI 2006 or WIV that was formulated with N3 alone. Additional groups were immunized with WIV and N3 adjuvant combined with pFliC(-gly). Homo and heterotypic humoral anti-WIV immune responses were assayed from serum and lung by ELISA and hemagglutination inhibition assay. Homo and heterotypic cellular immune responses to WIV and Influenza A NP were also determined. Results: WIV combined with N3 lipid adjuvant the pFliC(-gly) significantly increased homotypic influenza specific serum antibody responses (>200-fold), increased the IgG2 responses, indicating a mixed Th1/Th2-type immunity, and increased the HAI-titer (>100-fold). Enhanced cell-mediated IFNγ secreting influenza directed CD4+ and CD8+ T cell responses (>40-fold) to homotypic and heterosubtypic influenza A virus and peptides. Long-term and protective immunity was obtained. Conclusions: These results indicate that inactivated influenza virus that was formulated with N3 cationic adjuvant significantly enhanced broad systemic and mucosal influenza specific immune responses. These responses were broadened and further increased by incorporating DNA plasmids encoding FliC from S. typhimurum as an adjuvant providing long lasting protection against heterologous Influenza A/H1N1/CA09pdm virus challenge.

Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro.

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.

DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant.

Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

Antiretroviral therapy does not induce HIV type 1-specific neutralizing activity against autologous HIV type 1 isolates.

To determine the ability of antiretroviral treatment (ART) to deter viral replication in the long term, we tested autologous neutralization of HIV-1 primary isolates in sera from six chronically HIV-1-infected patients before and during such treatment. For comparison, heterologous neutralization was tested in the same samples by using a panel of eight primary HIV-1 isolates. Preceding ART, none of the patients' samples contained neutralizing antibodies against autologous HIV-1 isolates. Subsequently, despite successful ART treatment during a 12- to 19-month period and a rise in CD4 T cell counts, the patients' viral neutralizing capacity did not increase significantly. Furthermore, the partial heterologous neutralizing response was not improved in these patients. This outcome signifies the failure of an HIV-1-specific humoral immune response to improve, despite successful ART. Therefore, our results emphasize the need for immunologic intervention before cessation of ART in chronically HIV-1-infected patients to achieve sustainable control of viral replication.

A novel DNA adjuvant, N3, enhances mucosal and systemic immune responses induced by HIV-1 DNA and peptide immunizations.

AIMS: The study was designed to evaluate a novel cationic lipid DNA adjuvant (N3) and its function for HIV-1gp160/rev DNA plasmid delivered intranasally. The primary N3/HIV-DNA plasmid immunizations were boosted intranasally with a gp41 peptide in a anionic L3 adjuvant. This novel prime-boost strategy of mucosal immunization provided a broad HIV-1 envelope specific immunity, and recognition of viruses of subtypes A, B and C. CONCLUSIONS: Intranasal N3-adjuvanted gp160/rev DNA prime followed by one L3-peptide boosting immunization, induced broadly neutralizing antibodies against HIV-1 in the mucosa and systemically. The needle-free intranasal prime-boost strategy using two different adjuvant formulations reduced significantly the dose of DNA needed.

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity.

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.

Serum IgA of HIV-exposed uninfected individuals inhibit HIV through recognition of a region within the alpha-helix of gp41.

BACKGROUND: HIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro. OBJECTIVES: To analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals. METHODS: Sera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication. RESULTS: IgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582-588 (QARILAV) and corresponded to the leucine zip motif in the alpha-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5. CONCLUSION: These results have important implications for the development of vaccines and therapeutical strategies against HIV infection.

Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects.

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.