An Analysis of Linker-Dependent Effects on the APC Activation and In Vivo Immunogenicity of an R848-Conjugated Influenza Vaccine.

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.

Viral immunity: Basic mechanisms and therapeutic applications-a Keystone Symposia report.

Viruses infect millions of people each year. Both endemic viruses circulating throughout the population as well as novel epidemic and pandemic viruses pose ongoing threats to global public health. Developing more effective tools to address viruses requires not only in-depth knowledge of the virus itself but also of our immune system's response to infection. On June 29 to July 2, 2022, researchers met for the Keystone symposium "Viral Immunity: Basic Mechanisms and Therapeutic Applications." This report presents concise summaries from several of the symposium presenters.

Analysis of R848 as an Adjuvant to Improve Inactivated Influenza Vaccine Immunogenicity in Elderly Nonhuman Primates.

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.

Neuraminidase-specific antibody responses are generated in naive and vaccinated newborn nonhuman primates following virus infection.

Individuals younger than 6 months of age are at significant risk from influenza virus infection; however, there is currently no vaccine approved for this age group. Influenza virus neuraminidase (NA) has emerged as a potential additional target for vaccine strategies. In this study, we sought to understand the ability of newborns to mount an antibody response to NA. Here we employed a nonhuman primate model, given the similarities to humans in immune system and development. We measured antibody to NA following infection with an H1N1 virus or following vaccination and challenge. Administration of an inactivated virus vaccine was not capable of eliciting detectable NA-specific antibody, even in the presence of adjuvants previously shown to increase total virus-specific IgG. However, both naive and vaccinated newborns generated a NA-specific antibody response following virus infection. Interestingly, the presence of the vaccine-induced response did not prevent generation of systemic antibody to NA following challenge, although the respiratory response was reduced in a significant portion of newborns. These findings are the first, to our knowledge, to evaluate the newborn response to the influenza NA protein as well as the impact of previous vaccination on generation of these antibodies following virus infection.

Challenges for the Newborn Immune Response to Respiratory Virus Infection and 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.

TCR Dependent Metabolic Programming Regulates Autocrine IL-4 Production Resulting in Self-Tuning of the CD8+ T Cell Activation Setpoint.

The ability of T cells to sense and respond to environmental cues by altering their functional capabilities is critical for a safe and optimally protective immune response. One of the important properties that contributes to this goal is the activation set-point of the T cell. Here we report a new pathway through which TCR transgenic OT-I CD8+ T cells can self-tune their activation threshold. We find that in the presence of a strong TCR engagement event there is a shift in the metabolic programming of the cell where both glycolysis and oxidative phosphorylation are significantly increased. This diverges from the switch to a predominantly glycolytic profile that would be predicted following naïve T cell activation. Our data suggest this altered metabolic program results in the production of autocrine IL-4. Both metabolic pathways are required for this cytokine to be made. IL-4 signaling in the activated OT-I CD8+ T cell results in modulation of the sensitivity of the cell, establishing a higher activation setpoint that is maintained over time. Together these data demonstrate a novel mechanism for the regulation of IL-4 production in CD8+ T cells. Further, they reveal a new pathway for the self-tuning of peptide sensitivity. Finally, these studies uncover an unexpected role for oxidative phosphorylation in regulating differentiation in these cells.