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A number of studies have demonstrated the role of sex in regulating immune responses to vaccination. However, these findings have been limited to adults for both human and animal models. As a result, our understanding of the impact of sex on vaccine responses in the newborn is highly limited. Here, we probe this important question using a newborn non-human primate model. We leveraged our prior analysis of two cohorts of newborns, with one being mother-reared and one nursery-reared. This provided adequate numbers of males and females to interrogate the impact of sex on the response to inactivated influenza vaccines alone or adjuvanted with R848, flagellin, or both. We found that, in contrast to what has been reported in adults, the non-adjuvanted inactivated influenza virus vaccine induced similar levels of virus-specific IgG in male and female newborns. However, the inclusion of R848, either alone or in combination with flagellin, resulted in higher antibody titers in females compared to males. Sex-specific increases in the neutralizing antibody were only observed when both R848 and flagellin were present. These data, generated in the highly translational NHP newborn model, provide novel insights into the role of sex in the immune response of newborns.

Elderly individuals are highly susceptible to developing severe outcomes as a result of influenza A virus (IAV) infection. This can be attributed to alterations that span the aged immune system, which also result in reduced responsiveness to the seasonal inactivated vaccine. Given the rapidly increasing number of individuals in this age group, it is imperative that we develop strategies that can better protect this population from IAV-associated disease. Based on our previous findings that the TLR7/8 agonist resiquimod (R848) could efficiently boost responses in the newborn, another population with decreased vaccine responsiveness, we evaluated this adjuvant in an elderly African green monkey (AGM) model. AGM aged 16–24 years old (equivalent to 64–96 in human years) were primed and boosted with inactivated A/PuertoRico/8/1934 (H1N1) (IPR8) alone or directly linked to R848 (IPR8-R848). We observed increases in the level of circulating virus-specific IgM antibody 10 days following primary vaccination in AGM that were vaccinated with IPR8-R848, but not IPR8 alone. In addition, there were significant increases in virus-specific IgG after boosting selectively in the IPR8-R848 vaccinated animals. These findings provide insights into the ability of R848 to modulate the aged immune system in the context of IAV vaccination.

The initial months of life reflect an extremely challenging time for newborns as a naïve immune system is bombarded with a large array of pathogens, commensals, and other foreign entities. In many instances, the immune response of young infants is dampened or altered, resulting in increased susceptibility and disease following infection. This is the result of both qualitative and quantitative changes in the response of multiple cell types across the immune system. Here we provide a review of the challenges associated with the newborn response to respiratory viral pathogens as well as the hurdles and advances for vaccine-mediated protection.

Subunit or inactivated vaccines comprise the majority of vaccines used against viral and bacterial pathogens. However, compared to their live/attenuated counterparts, these vaccines often demonstrate reduced immunogenicity, requiring multiple boosters and or adjuvants to elicit protective immune responses. For this reason, studies of adjuvants and the mechanism through which they can improve inactivated vaccine responses are critical for the development of vaccines with increased efficacy. Studies have shown that the direct conjugation of adjuvant to antigen promotes vaccine immunogenicity, with the advantage of both the adjuvant and antigen targeting the same cell. Using this strategy of direct linkage, we developed an inactivated influenza A (IAV) vaccine that is directly conjugated with the Toll-like receptor 7/8 agonist resiquimod (R848) through a heterobifunctional crosslinker. Previously, we showed that this vaccine resulted in improved protection and viral clearance in newborn nonhuman primates compared to a non-adjuvanted vaccine. We subsequently discovered that the choice of linker used to conjugate R848 to the virus alters the stimulatory activity of the vaccine, promoting increased maturation and proinflammatory cytokine production from DC differentiated in vitro. With this knowledge, we explored how the choice of crosslinker impacts the stimulatory activity of these vaccines. We found that the linker choice alters signaling through the NF-κB pathway in human monocyte-derived dendritic cells (moDCs). Further, we extended our analyses to in vivo differentiated APC present in human peripheral blood, replicating the linker-dependent differences found in in vitro differentiated cells. Finally, we demonstrated in a mouse model that the choice of linker impacts the amount of IAV-specific IgG antibody produced in response to vaccination. These data enhance our understanding of conjugation approaches for improving vaccine immunogenicity.