An R848-Conjugated Influenza Virus Vaccine Elicits Robust Immunoglobulin G to Hemagglutinin Stem in a Newborn Nonhuman Primate Model.

Eliciting broadly protective antibodies is a critical goal for the development of more effective vaccines against influenza. Optimizing protection is of particular importance in newborns, who are highly vulnerable to severe disease following infection. An effective vaccination strategy for this population must surmount the challenges associated with the neonatal immune system as well as mitigate the inherent immune subdominance of conserved influenza virus epitopes, responses to which can provide broader protection. Here, we show that prime-boost vaccination with a TLR7/8 agonist (R848)-conjugated influenza A virus vaccine elicits antibody responses to the highly conserved hemagglutinin stem and promotes rapid induction of virus neutralizing stem-specific antibodies following viral challenge. These findings support the efficacy of R848 as an effective adjuvant for newborns and demonstrate its ability to enhance antibody responses to subdominant antigenic sites in this at-risk population.

An R848 adjuvanted influenza vaccine promotes early activation of B cells in the draining lymph nodes of non-human primate neonates.

Impaired immune responsiveness is a significant barrier to vaccination of neonates. By way of example, the low seroconversion observed following influenza vaccination has led to restriction of its use to infants over 6 months of age, leaving younger infants vulnerable to infection. Our previous studies using a non-human primate neonate model demonstrated that the immune response elicited following vaccination with inactivated influenza virus could be robustly increased by inclusion of the Toll-like receptor agonist flagellin or R848, either delivered individually or in combination. When delivered individually, R848 was found to be the more effective of the two. To gain insights into the mechanism through which these adjuvants functioned in vivo, we assessed the initiation of the immune response, i.e. at 24 hr, in the draining lymph node of neonate non-human primates. Significant up-regulation of co-stimulatory molecules on dendritic cells could be detected, but only when both adjuvants were present. In contrast, R848 alone could increase the number of cells in the lymph node, presumably through enhanced recruitment, as well as B-cell activation at this early time-point. These changes were not observed with flagellin and the dual adjuvanted vaccine did not promote increases beyond those observed with R848 alone. In vitro studies showed that R848 could promote B-cell activation, supporting a model wherein a direct effect on neonate B-cell activation is an important component of the in vivo potency of R848 in neonates.

Sex-Dependent Effects on Influenza-Specific Antibody Quantity and Neutralizing Activity following Vaccination of Newborn Non-Human Primates Is Determined by Adjuvants.

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.

TLR agonists induce sustained IgG to hemagglutinin stem and modulate T cells following newborn vaccination.

The newborn immune system is characterized by diminished immune responses that leave infants vulnerable to virus-mediated disease and make vaccination more challenging. Optimal vaccination strategies for influenza A virus (IAV) in newborns should result in robust levels of protective antibodies, including those with broad reactivity to combat the variability in IAV strains across seasons. The stem region of the hemagglutinin (HA) molecule is a target of such antibodies. Using a nonhuman primate model, we investigate the capacity of newborns to generate and maintain antibodies to the conserved stem region following vaccination. We find adjuvanting an inactivated vaccine with the TLR7/8 agonist R848 is effective in promoting sustained HA stem-specific IgG. Unexpectedly, HA stem-specific antibodies were generated with a distinct kinetic pattern compared to the overall response. Administration of R848 was associated with increased influenza-specific T follicular helper cells as well as Tregs with a less suppressive phenotype, suggesting adjuvant impacts multiple cell types that have the potential to contribute to the HA-stem response.

Understanding Antibody Responses in Early Life: Baby Steps towards Developing an Effective Influenza Vaccine.

The immune system of young infants is both quantitatively and qualitatively distinct from that of adults, with diminished responsiveness leaving these individuals vulnerable to infection. Because of this, young infants suffer increased morbidity and mortality from respiratory pathogens such as influenza viruses. The impaired generation of robust and persistent antibody responses in these individuals makes overcoming this increased vulnerability through vaccination challenging. Because of this, an effective vaccine against influenza viruses in infants under 6 months is not available. Furthermore, vaccination against influenza viruses is challenging even in adults due to the high antigenic variability across viral strains, allowing immune evasion even after induction of robust immune responses. This has led to substantial interest in understanding how specific antibody responses are formed to variable and conserved components of influenza viruses, as immune responses tend to strongly favor recognition of variable epitopes. Elicitation of broadly protective antibody in young infants, therefore, requires that both the unique characteristics of young infant immunity as well as the antibody immunodominance present among epitopes be effectively addressed. Here, we review our current understanding of the antibody response in newborns and young infants and discuss recent developments in vaccination strategies that can modulate both magnitude and epitope specificity of IAV-specific antibody.

Influenza-infected newborn and adult monkeys exhibit a strong primary antibody response to hemagglutinin stem.

The specificity of antibodies (Abs) generated against influenza A virus (IAV) infection can significantly alter protection and viral clearance. At present, the impact of age upon this process is relatively unexplored. Here, we evaluated the Ab response in newborn and adult African green monkeys following infection with IAV using a strain that enables us to determine the immunodominance (ID) hierarchy of the Ab response to hemagglutinin (HA), the principal target of protective Abs. This revealed altered ID patterns in the early IgM anti-HA response in newborns versus adults that converged over time. While the IgG ID profiles for HA in newborn and adult monkeys were similar, this was not the case for IgA. Importantly, HA stem-specific Abs were generated robustly and similarly in newborns and adults in terms of quality and quantity. Together, these results demonstrate that newborns and adults can differ in the Ab ID pattern established following infection and that the ID pattern can vary across isotypes. In addition, newborns have the ability to generate potent HA stem-specific Ab responses. Our findings further the understanding of the newborn response to IAV antigens and inform the development of improved vaccines for this at-risk population.

The choice of linker for conjugating R848 to inactivated influenza virus determines the stimulatory capacity for innate immune cells.

Inactivated influenza vaccines are not approved for use in infants less than 6 months of age due to poor immunogenicity in that population. While the live attenuated influenza vaccine has the potential to be more immunogenic, it is not an option for infants and other vulnerable populations, including the elderly and immunocompromised individuals due to safety concerns. In an effort to improve the immunogenicity of the inactivated vaccine for use in vulnerable populations, we have used an approach of chemically crosslinking the Toll-like receptor (TLR) 7/8 agonist R848 directly to virus particles. We have reported previously that an R848-conjugated, inactivated vaccine is more effective at inducing adaptive immune responses and protecting against lung pathology in influenza challenged neonatal African green monkeys than is the unmodified counterpart. In the current study, we describe a second generation vaccine that utilizes an amide-sulfhydryl crosslinker with different spacer chemistry and length to couple R848 to virions. The new vaccine has significantly enhanced immunostimulatory activity for murine macrophages and importantly for monocyte derived human dendritic cells. Demonstration of the significant differences in stimulatory activity afforded by modest changes in linker impacts our fundamental view of the design of TLR agonist-antigen vaccines.

Effects of a single dose of oral iron on hepcidin concentrations in human urine and serum analyzed by a robust LC-MS/MS method.

BACKGROUND: The measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. METHODS: In order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8 h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease. RESULTS: The linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r(2)=0.9512±0.0202 (n=5) for urine and r(2)=0.9709±0.0291 (n=5) for serum [r(2)=mean±SD]. Compared to baseline, the levels of urinary hepcidin between 2-4 h and 4-8 h of both women and men showed significant increases with p<0.05 and p<0.001, respectively. The levels of serum hepcidin between 4 h and 8 h in both women and men showed significant increases, compared with baseline values, with both p<0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points. CONCLUSIONS: We have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men.