Intranasal Live Influenza Vaccine Priming Elicits Localized B Cell Responses in Mediastinal Lymph Nodes

Abstract

Pandemic live attenuated influenza vaccines (pLAIV) prime subjects for a robust neutralizing antibody response upon subsequent administration of a pandemic inactivated subunit vaccine (pISV). However, a difference was not detected in H5-specific memory B cells in the peripheral blood between pLAIV-primed and unprimed subjects prior to pISV boost. To investigate the mechanism underlying pLAIV priming, we vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LN, spleen, and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination. We have previously shown that pandemic live attenuated influenza vaccines (pLAIV) prime for a rapid and robust antibody response on subsequent administration of inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody (Ab) responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a nonhuman primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We show that the nonhuman primate model recapitulates the serologic observations from clinical studies. Further, we found that pLAIVs induced robust germinal center B cell responses in the mediastinal lymph node. Subsequent boosting with pISV in pLAIV-primed animals resulted in detection of B cells in the axillary lymph nodes, spleen, and peripheral blood. We demonstrate that intranasally administered pLAIV elicits a highly localized germinal center B cell response in the mediastinal lymph node that is rapidly recalled following pISV boost into germinal center reactions at numerous distant immune sites.

Hemagglutinin head-specific responses dominate over stem-specific responses following prime boost with mismatched vaccines

“…Using a fluorescent stabilized stem immunogen (27), we determined that most, if not all, H5 + H1 + double-positive cells bound the HA stem while the H5 + single-positive cells did not (Supplemental Figure 1B). AGMs vaccinated with H5N1 pLAIV showed a small but detectable population of H5 + H1 + double-positive stem-specific memory B cells in the peripheral blood ( Figure 1, A and B) and MLNs at day 14 ( Figure 1C), but, as previously reported (21), the frequency of H5 + head-specific memory B cells was markedly higher in both the peripheral blood and MLNs at day 14 after pLAIV (0.05%-0.19% and 0.21%-0.64% for stem-and head-specific frequencies in the MLN, respectively). Consistent with these findings and our previous reports (12,21), serum-neutralizing Abs were not detected at days 14 and 28 against either A/Vietnam/1203/04 (H5N1) or A/California/07/09 (H1N1pdm09) viruses following administration of H5N1 pLAIV (Table 1).…”

“…Exploring the immunologic basis for these clinical observations in AGMs, we recently reported that intranasal administration of H5N1 pLAIV followed by matched pIIV boost generates a robust H5-specific B cell response and H5-specific neutralizing Ab response. Further, we found that H5-specific memory B cells are generated in the MLN following pLAIV (21). We did not, however, assess the ability of pLAIV to generate a broad stem-specific memory B cell pool that can be recalled upon pIIV boost.…”

“…We recently established an African green monkey (AGM) IAV model and established that vaccination recapitulates the clinical phenomena observed during prime-boost human vaccination with pLAIV-pIIV (12,21). The AGM model allowed us to dissect the HA-specific Ab and B cell responses to matched pLAIV-pIIV vaccination in the peripheral blood and lymphoid tissues.…”

Humanized Mice for Live-Attenuated Vaccine Research: From Unmet Potential to New Promises

“…A more comprehensive (although not exhaustive) summary of LAV attenuation mechanisms is provided in Table 2, and is associated with the following additional references: . [120,121] n/a (reassortant vaccine) [191,192] Mouse [194]; NHP [195]; Pig [196] Historical aspects of live-attenuated vaccine (LAV) creation, such as method of generation, as well as the date of the pathogen's isolation or attenuation are listed. Mechanisms of LAV attenuation are categorized into (i) genetic similarity to their parental strain or a virulent strain (with the exception of smallpox and influenza LAVs), (ii) studies on mechanisms governing LAV attenuation performed in vitro, and (iii) animal models used for in vivo investigation.…”

“…Additional strategies employing HIV-1 related viruses [210,211] or engineered viruses harboring HIV-1 or SIV antigens [212,213] have also been tested in NHPs. NHPs have also been employed to investigate and/or evaluate the immunogenicity of many other LAVs, such as viral vectors harboring Ebola virus glycoproteins [214][215][216][217][218], LAIV [195], OPV [189,190], MV-LAV [150,185,197,[219][220][221], BCG [156], live-attenuated Venezuelan equine encephalitis virus [222], YFV-17D [151] or live-attenuated Zika virus [43]. NHP models for LAV research, however, present significant limitations.…”