squalene and Orthomyxoviridae-Infections

Poor immune responses to inactivated influenza vaccine can be improved by effective and safe adjuvants to increase antibody titers and cellular protective response. In our study, AddaVax and PolyI:C combined adjuvant (AP adjuvant) were used for influenza vaccine development. After immunizing BALB/c mice and Wistar rats intramuscularly, Split inactivated H3N2 vaccine adjuvanted with AP elicited higher serum hemagglutination-inhibition antibodies and IgG titers. We demonstrated that AP induced a transient innate immune cytokines production at the injection site, induced H3N2 uptake by DCs, increased recruitment of monocytes and DCs in LNs, and promoted H3N2 vaccine migration; AP facilitated vaccines to induce a vigorous adaptive immune response. Besides, AP showed good safety as shown by lymph nodes (LNs) size, spleens index of BALB/c mice, and weight changes and C-reaction protein level of BALB/c mice and Wistar rats after repeated administration of high-dose vaccine with or without adjuvant. These findings indicate that AP is a potential novel adjuvant and can be used as a safe and effective adjuvant for MDCK-based influenza inactivated vaccine to induce cellular and antibody protective response.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Immunity; Influenza A Virus, H3N2 Subtype; Influenza Vaccines; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Polysorbates; Rats; Rats, Wistar; Squalene

The novel H7N9 avian influenza A virus has caused human infections in China since 2013; some isolates from the fifth wave of infections have emerged as highly pathogenic avian influenza viruses. Recombinant hemagglutinin proteins of H7N9 viruses can be rapidly and efficiently produced with low-level biocontainment facilities. In this study, recombinant H7 antigen was obtained from engineered stable clones of Chinese Hamster Ovary (CHO) cells for subsequent large-scale production. The stable CHO cell clones were also adapted to grow in serum-free suspension cultures. To improve the immunogenicity of the recombinant H7 antigens, we evaluated the use of a novel combination adjuvant of PELC and CpG (PELC/CpG) to augment the anti-H7N9 immune responses in mice. We compared the effects with other adjuvants such as alum, AddaVax (MF59-like), and several Toll-like receptor ligands such as R848, CpG, and poly (I:C). With the PELC/CpG combination adjuvant, CHO cell-expressed rH7 antigens containing terminally sialylated complex type N-glycans were able to induce high titers of neutralizing antibodies in sera and conferred protection following live virus challenges. These data indicate that the CHO cell-expressed recombinant H7 antigens and a PELC/CpG combination adjuvant can be used for H7N9 subunit vaccine development.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Neutralizing; Antibodies, Viral; Cell Line; CHO Cells; CpG Islands; Cricetulus; Female; Hemagglutination Inhibition Tests; Hemagglutinin Glycoproteins, Influenza Virus; Hemagglutinins; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Mice; Mice, Inbred BALB C; Orthomyxoviridae Infections; Polysorbates; Recombinant Proteins; Squalene; Vaccines, Subunit

Carbohydrate fatty acid sulphate esters (CFASEs) formulated in a squalane-in-water emulsion are effective adjuvants for humoral responses to a wide range of antigens in various animal species but rise in body temperature and local reactions albeit mild or minimal hampers application in humans. In rabbits, body temperature increased 1°C one day after intramuscular (IM) injection, which returned to normal during the next day. The effect increased with increasing dose of CFASE but not with the number of injections (up to 5). Antigen enhanced the rise in body temperature after booster immunization (P<0.01) but not after priming. Synthetic CFASEs are mixtures of derivatives containing no sulphate, one or multiple sulphate groups and the monosulphate derivatives (CMS) were isolated, incorporated in a squalane in-water emulsion and investigated. In contrast to CFASE, CMS adjuvant did not generate rise in body temperature or local reactions in rabbits immunized with a purified, recombinant malaria chimeric antigen R0.10C. In comparison to alum, CMS adjuvant revealed approximately 30-fold higher antibody titres after the first and >100-fold after the second immunization. In ferrets immunized with 7.5μg of inactivated influenza virus A/H7N9, CMS adjuvant gave 100-fold increase in HAI antibody titres after the first and 25-fold after the second immunisation, which were 10-20-fold higher than with the MF59-like AddaVax adjuvant. In both models, a single immunisation with CMS adjuvant revealed similar or higher titres than two immunisations with either benchmark, without detectable systemic and local adverse effects. Despite striking chemical similarities with monophospholipid A (MPL), CMS adjuvant did not activate human TLR4 expressed on HEK cells. We concluded that the synthetic CMS adjuvant is a promising candidate for poor immunogens and single-shot vaccines and that rise in body temperature, local reactions or activation of TLR4 is not a pre-requisite for high adjuvanticity.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Viral; Body Temperature; Carbohydrates; Drug Compounding; Esters; Fatty Acids; Ferrets; HEK293 Cells; Hemagglutination Inhibition Tests; Humans; Immunity, Humoral; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Influenza, Human; Injections, Intramuscular; Lipid A; Orthomyxoviridae Infections; Polysorbates; Rabbits; Squalene; Toll-Like Receptor 4; Vaccination

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 μg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)3 or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)3 or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 μg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 μg to 0.5 μg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.

Topics: Adjuvants, Immunologic; Aluminum Hydroxide; Animals; Cross Reactions; Female; Hemagglutination Inhibition Tests; Immunity, Humoral; Influenza A Virus, H7N7 Subtype; Influenza A Virus, H7N9 Subtype; Influenza Vaccines; Mice, Inbred BALB C; Neutralization Tests; Orthomyxoviridae Infections; Polysorbates; Squalene; Survival Analysis; Vaccines, Inactivated; Vaccines, Subunit

The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C), and an RNA hairpin derived from Sendai virus (SeV) Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C) also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibodies, Viral; Dogs; Emulsions; Enzyme-Linked Immunosorbent Assay; Female; HEK293 Cells; Hemagglutinin Glycoproteins, Influenza Virus; Humans; Immunization; Influenza A virus; Influenza Vaccines; Injections, Intramuscular; Madin Darby Canine Kidney Cells; Mice; Mice, Inbred BALB C; Nucleic Acid Conformation; Orthomyxoviridae Infections; Poly I-C; Polysorbates; Recombinant Fusion Proteins; RNA, Viral; Sendai virus; Squalene