Surviving a single infection often results in lifelong immunity to the infecting pathogen. Such protection is mediated, in large part, by two main B cell memory 'walls'-namely , longlived plasma cells and memory B cells. The cellular and molecular processes that drive the production of long-lived plasma cells and memory B cells are subjects of intensive research and have important implications for global health. Indeed, although nearly all vaccines in use today depend on their ability to induce B cell memory , we have not yet succeeded in developing vaccines for some of the world's most deadly diseases, including AIDS and malaria. Here, we describe the two-phase process by which antigen drives the generation of long-lived plasma cells and memory B cells and highlight the challenges for successful vaccine development in each phase.

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Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17–36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4′-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500–12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25–1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.

We show that minor capsid protein L2 is full length in clinical virion isolates and prepare furin-cleaved pseudovirus (fcPsV) as a model of the infectious intermediate for multiple human papillomavirus (HPV) types. These fcPsV do not require furin for in vitro infection, and are fully infectious in vivo. Both the γ-secretase inhibitor XXI and carrageenan block fcPsV infection in vitro and in vivo implying that they act after furin-cleavage of L2. Despite their enhanced exposure of L2 epitopes, vaccination with fcPsV particles fails to induce L2 antibody, although L1-specific responses are similar to PsV with intact L2. FcPsV can be applied in a simple, high-throughput neutralization assay that detects L2-specific neutralizing antibodies with >10-fold enhanced sensitivity compared with the PsV-based assay. The PsV and fcPsV-based assays exhibit similar sensitivity for type-specific antibodies elicited by L1 virus-like particles (VLP), but the latter improves detection of L1-specific cross-type neutralizing antibodies.

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected based on their ability to bind, gather and present antigen to T follicular helper (Tfh) cells. Here we show that human GC B cells have intrinsically higher affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared to naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen-affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structure was dictated by the intrinsic antigen-affinity thresholds of GC B cells. Low affinity antigens triggered continuous engagement and dis-engagement of membrane associated antigens whereas high affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

We sought to define the protective epitopes within the amino terminus of human papillomavirus (HPV) type 16 minor capsid protein L2. Passive transfer of mice with rabbit antisera to HPV16 L2 peptides 17–36, 32–51 and 65–81 provided significant protection against vaginal HPV16 challenge, whereas antisera to 47–66, 108–120 or 373–392 did not. Vaccination with L1 virus-like particles induces a high titer, but generally type-restricted neutralizing antibody response. Conversely, vaccination with L2 11–88, especially multimers thereof, induces antibodies that neutralize a broad range of papillomavirus types, albeit at lower titers than for L1 VLP. With the intent of enhancing the immunogenicity and the breadth of protection by focusing the immune response to the key protective epitopes, we designed L2 fusion proteins consisting of residues ∼11–88 of eight divergent mucosal HPV types 6, 16, 18, 31, 39, 51, 56, 73 (11–88×8) or residues ∼13–47 of fifteen HPV types (13–47×15). The 11–88×8 was significantly more immunogenic than 13–47×15 in Balb/c mice regardless of the adjuvant used, suggesting the value of including the 65–81 protective epitope in the vaccine. Since the L2 47–66 peptide antiserum failed to elicit significant protection, we generated an 11–88×8 construct deleted for this region in each subunit (11–88×8Δ). Mice were vaccinated with 11–88×8 and 11–88×8Δ to determine if deletion of this non-protective epitope enhanced the neutralizing antibody response. However, 11–88×8Δ was significantly less immunogenic than 11–88×8, and even the addition of a known T helper epitope, PADRE, to the construct (11–88×8ΔPADRE) failed to recover the immunogenicity of 11–88×8 in C57BL/6 mice, suggesting that while L2 47–66 is not a critical protective or T helper epitope, it nevertheless contributes to the immunogenicity of the L2 11–88×8 multimer vaccine.

Immunization with L1 as pentavalent capsomeres or virus-like particles (VLPs) generates high and long-lived titers of neutralizing antibodies and protection primarily against the human papillomavirus (HPV) type from which the vaccine was derived. Conversely, vaccination with L2 minor capsid protein derived from multiple HPV types induces lower titer, but more broadly neutralizing and protective antibody responses. We combined the advantages of each protective antigen by immunization with titrated doses of multi-type L2 with either L1 capsomeres or VLP. We observed no significant interference between the L1 and L2 antibody response upon coadministration of L1 vaccines with multi-type L2 vaccines. BACKGROUNDHPV is responsible for 5% of all cancers worldwide, including cervical cancer and the majority of vaginal, vulval, penile, anal and a subset of certain head and neck cancers [1]. Although HPV16 and HPV18 cause 50% and 20% of cervical cancer cases respectively, there are more than a dozen other 'oncogenic' types of genital HPV [2]. Long term protection against all oncogenic types through vaccination is necessary to eventually eliminate cervical cancer and the need for expensive screening programs [3][4][5]. Other benign HPV types are responsible for considerable morbidity, including genital warts associated primarily with HPV6 and HPV11 infections. The currently licensed vaccines, Cervarix and Gardasil, are derived from major capsid protein L1 virus-like particles self assembled in insect or yeast cells respectively [6,7]. These vaccines both target the two most important oncogenic HPV types, HPV16 and HPV18, although Gardasil also contains HPV6 and HPV11 L1 VLP to protect against benign genital warts. The current vaccines do not Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptVaccine. Author manuscript; available in PMC 2011 June 17. Immunization with L1 VLPs generates high titer serum neutralizing antibodies that are primarily type-specific, although limited cross-reactivity with the additional oncogenic types associated with cervical cancer has been observed [9,10]. L1 VLP are protective even without an adjuvant [11][12][13][14], but the current vaccines both are formulated in aluminum salts (amorphous aluminum hydroxyphosphate sulfate in Gardasil and aluminum hydroxide in Cervarix), and Cervarix also includes the TLR4 agonist monophosphoryl lipid A (MPL), presumably with the goal of enhancing cross-neutralization of closely related types and sustaining the neutralizing antibody response [3]. The licensed L1 VLP vaccines provi...

Depending upon viral genotype, productive papillomavirus infection and disease display preferential tropism for cutaneous or mucosal stratified squamous epithelia, although the mechanisms are unclear. To investigate papillomavirus entry tropism, we used reporter pseudovirions based on various cutaneous and mucosal papillomavirus species, including the recently identified murine papillomavirus. Pseudovirus transduction of BALB/c mice was examined using an improved murine skin infection protocol and a previously developed cervicovaginal challenge model. In the skin, HPV5, HPV6, HPV16, BPV1 and MusPV1 pseudovirions preferentially transduced keratinocytes at sites of trauma, similar to the genital tract. Skin infection, visualized by in vivo imaging using a luciferase reporter gene, peaked between days 2–3 and rapidly diminished for all pseudovirion types. Murine cutaneous and genital tissues were similarily permissive for pseudovirions of HPV types 5,6,8,16,18,26,45,51,58 and animal papillomaviruses BPV1 and MusPV1, implying that papillomavirus’ tissue and host tropism is governed primarily by post-entry regulatory events in the mouse.