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Autophagy is a cellular degradation process with an increasingly recognised importance in many biological pathways such as nutrient sensing, stress responses and development. We present a straightforward assay for autophagy which combines the sensitivity of the EGFP-LC3 reporter protein with the throughput capacity and quantitative power of flow cytometry. Because saponin extraction is specific for the non-autophagosome associated EGFP-LC3-I form of the protein, flow cytometry can be used to measure total fluorescence of saponin extracted HOS-EGFP-LC3 cells as a measure of the levels of autophagosome associated EGFP-LC3-II. Combined with inhibitors of degradation, we have adapted this assay to differentiate between constitutive and induced autophagy and to quantify the changes in flux of the system. Moreover, using direct antibody staining for the endogenous LC3 protein, we have extended this assay to the detection of autophagosome formation in non-transfected cells.

Purpose of review To summarize recent progress in the development of adjuvants with a special focus on adjuvants that enhance B-cell responses to protein-based vaccines. Both established and new experimental approaches are described and also briefly we discuss how adjuvants and virus-based vaccines interact with the immune system. Recent findings Two new adjuvants were recently approved for human applications and many others are in preclinical or clinical testing. Significant advances were made to describe the mechanism of action of adjuvants. For example, aluminum hydroxide salts were shown to engage Nalp3, a member of the cytosolic NOD-like receptors and activation of B cells via invariant natural killer cell presentation of alpha-galactosylceramide was described. The effects of Toll-like receptor ligands on B-cell differentiation were further characterized and a peptide derived from IPS-1, a cytosolic signaling molecule, was shown to provide adjuvant effect. Stimulation of protective antibodies against HIV-1 may require extensive antibody affinity maturation, thus long-term exposure or repeated administration of antigen may be needed to induce effective B-cell responses. Summary Advances in our understanding of how specific signaling pathways link innate and adaptive immunity provides a basis for the design of improved adjuvants to promote broad and potent B-cell responses.

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.

Selected TLR ligands are under evaluation as vaccine adjuvants and are known to activate dendritic cells (DCs) and B cells to affect vaccine-induced Ab responses. However, the relative contribution of the two main human DC subsets, myeloid (MDCs) and plasmacytoid (PDCs), in supporting B cell responses to TLR ligands remains poorly understood. We found that PDCs but not MDCs markedly enhanced B cell proliferation in response to TLR7/8-L, an imidazoquinoline derivative, and to a lesser extent to TLR9 ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-L-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. In response to TLR7/8 stimulation, PDCs mediated the up-regulation of transcription factors B lymphocyte-induced maturation protein 1 and X-box binding protein 1 and enhanced differentiation of B cells into IgM-, IgG-, and IgA-producing cells. Type I IFN produced to high levels by PDCs was the principal mediator of the effects on TLR7/8 stimulation. Although MDCs expressed higher levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system. These data help decipher the different roles of PDCs and MDCs for modulating human B cell responses and can contribute to selection of specific TLR ligands as vaccine adjuvants.

The host type I interferon (IFN) system is central in antiviral defence and represents one of the greatest obstacles for a virus to overcome in order to establish a productive infection. Viruses have evolved many different mechanisms to repress the effects of the type I IFN system. For example, a number of viruses encode viral proteases, which can directly cleave and inactivate key components of the type I IFN induction and signalling pathway. Others recruit the ubiquitin proteasome system to destabilise proteins that are important for IFN responses. There are also many known viral proteins, which bind to and sequester proteins of the type I IFN system in an inactive state. Here, we review each of these different mechanisms of viral escape from the type I IFN response with examples from a range of viruses.

Most viral vaccines protect against disease by generating neutralizing antibodies. This Review examines the problem of eliciting broad HIV-1 neutralization through vaccination by drawing parallels with the successful subunit influenza virus vaccine and with efforts to develop a pandemic influenza vaccine. The ability to elicit broadly neutralizing antibody responses against HIV-1 is a crucial goal for a prophylactic HIV-1 vaccine. Here, we discuss the difficulties of achieving broad HIV-1 neutralization in the context of both the effective annual human influenza virus vaccine and the need to develop a pandemic influenza vaccine. Immunogen-design strategies are underway to target functionally conserved regions of the HIV-1 envelope glycoproteins, and similar strategies might be applicable to pandemic influenza virus vaccine development. Efforts to develop broadly neutralizing vaccines against either HIV-1 or influenza virus might establish a paradigm for future vaccines against highly variable pathogens.

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

The main pathway for the induction of type I interferons (IFN) by viruses is through the recognition of viral RNA by cytosolic receptors and the subsequent activation of interferon regulatory factor 3 (IRF-3), which drives IFN-α/β transcription. In addition to their role in inducing an antiviral state, type I IFN also play a role in modulating adaptive immune responses, in part via their effects on dendritic cells (DCs). Many viruses have evolved mechanisms to interfere with type I IFN induction, and one recently reported strategy for achieving this is by targeting IRF-3 for degradation, as shown for rotavirus nonstructural protein 1 (NSP1). It was therefore of interest to investigate whether rotavirus-exposed DCs would produce type I IFN and/or mature in response to the virus. Our results demonstrate that IRF-3 was rapidly degraded in rotavirus-infected mouse embryonic fibroblasts (MEFs) and type I IFN was not detected in these cultures. In contrast, rotavirus induced type I IFN production in myeloid DCs (mDCs), resulting in their activation. Type I IFN induction in response to rotavirus was reduced in mDCs from IRF-3−/−mice, indicating that IRF-3 was important for mediating the response. Exposure of mDCs to UV-treated rotavirus induced significantly higher type I IFN levels, suggesting that rotavirus-encoded functions also antagonized the response in DCs. However, in contrast to MEFs, this action was not sufficient to completely abrogate type I IFN induction, consistent with a role for DCs as sentinels for virus infection.

The human immunodeficiency virus (HIV-1) envelope glycoproteins (Env) mediate virus entry through a series of complex conformational changes triggered by binding to the receptors CD4 and CCR5/CXCR4. Broadly neutralizing antibodies that recognize conserved Env epitopes are thought to be an important component of a protective immune response. However, to date, HIV-1 Env immunogens that elicit broadly neutralizing antibodies have not been identified, creating hurdles for vaccine development. Small-molecule CD4-mimetic compounds engage the CD4-binding pocket on the gp120 exterior Env and induce Env conformations that are highly sensitive to neutralization by antibodies, including antibodies directed against the conserved Env region that interacts with CCR5/CXCR4. Here, we show that CD4-mimetic compounds sensitize primary HIV-1 to neutralization by antibodies that can be elicited in monkeys and humans within 6 months by several Env vaccine candidates, including gp120 monomers. Monoclonal antibodies directed against the gp120 V2 and V3 variable regions were isolated from the immunized monkeys and humans; these monoclonal antibodies neutralized a primary HIV-1 only when the virus was sensitized by a CD4-mimetic compound. Thus, in addition to their direct antiviral effect, CD4-mimetic compounds dramatically enhance the HIV-1-neutralizing activity of antibodies that can be elicited with currently available immunogens. Used as components of microbicides, the CD4-mimetic compounds might increase the protective efficacy of HIV-1 vaccines. IMPORTANCE Preventing HIV-1 transmission is a high priority for global health. Eliciting antibodies that can neutralize transmitted strains of HIV-1 is difficult, creating problems for the development of an effective vaccine. We found that small-molecule CD4-mimetic compounds sensitize HIV-1 to antibodies that can be elicited in vaccinated humans and monkeys. These results suggest an approach to prevent HIV-1 sexual transmission in which a virus-sensitizing microbicide is combined with a vaccine.

Due to the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing antibodies to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited antibody responses, we utilized single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques following five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third complementarity determining region (CDR3) of Ig heavy chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in antibody sequences isolated at late immunization time point compared to the early time point. Antibodies with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of antigen affinity selection in antibody maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high resolution understanding of the dynamically evolving CD4bs-specific B cell response following Env immunization in primates.

Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of broadly neutralizing antibodies (bNAbs) in HIV-1-infected individuals. However, the application of NGS to the preclinical assessment of human vaccines, particularly the monitoring of vaccine-induced B-cell responses in a nonhuman primate (NHP) model, has not been reported. Here, we present a longitudinal NGS analysis of memory B-cell responses to an HIV-1 trimer vaccine in a macaque that has been extensively studied by single B-cell sorting and antibody characterization. We first established an NHP antibodyomics pipeline using the available 454 pyrosequencing data from this macaque and developed a protocol to sequence the NHP antibody repertoire in an unbiased manner. Using these methods, we then analyzed memory B-cell repertoires at four time points of NHP immunization and traced the lineages of seven CD4-binding site (CD4bs)-directed monoclonal antibodies previously isolated from this macaque. Longitudinal analysis revealed distinct patterns of B-cell lineage development in response to an HIV-1 trimer vaccine. While the temporal B-cell repertoire profiles and lineage patterns provide a baseline for comparison with forthcoming HIV-1 trimer vaccines, the newly developed NHP antibody NGS technologies and antibodyomics tools will facilitate future evaluation of human vaccine candidates., IMPORTANCE The nonhuman primate model has been widely used in the preclinical assessment of human vaccines. Next-generation sequencing of B-cell repertoires provides a quantitative tool to analyze B-cell responses to a vaccine. In this study, the longitudinal B-cell repertoire analysis of a rhesus macaque immunized with an HIV-1 trimer vaccine revealed complex B-cell lineage patterns and showed the potential to facilitate the evaluation of future HIV-1 vaccines. The repertoire sequencing technologies and antibodyomics methods reported here can be extended to vaccine development for other human pathogens utilizing the nonhuman primate model.

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Isolation of mAbs elicited by vaccination provides opportunities to define the development of effective immunity. Ab responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens display narrow neutralizing activity with limited capacity to block infection by tier 2 viruses. Intense work in the field suggests that improved Env immunogens are forthcoming, and it is therefore important to concurrently develop approaches to investigate the quality of vaccine-elicited responses at a higher level of resolution. In this study, we cloned a representative set of mAbs elicited by a model Env immunogen in rhesus macaques and comprehensively characterized their genetic and functional properties. The mAbs were genetically diverse, even within groups of Abs targeting the same subregion of Env, consistent with a highly polyclonal response. mAbs directed against two subdeterminants of Env, the CD4 binding site and V region 3, could in part account for the neutralizing activity observed in the plasma of the animal from which they were cloned, demonstrating the power of mAb isolation for a detailed understanding of the elicited response. Finally, through comparative analyses of mAb binding and neutralizing capacity of HIV-1 using matched Envs, we demonstrate complex relationships between epitope recognition and accessibility, highlighting the protective quaternary packing of the HIV-1 spike relative to vaccine-induced mAbs.

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

Viruses typically elicit potent adaptive immune responses, and live-virus-based vaccines are among the most efficient human vaccines known. The mechanisms by which viruses stimulate adaptive immune responses are not fully understood, but activation of innate immune signaling pathways in the early phase of the infection may be of importance. In addition to stimulating immune responses to viral antigens expressed in infected cells, viruses can also provide adjuvant signals to coimmunized protein antigens. Using recombinant Semliki Forest virus (rSFV)-based vaccines, we show that rSFV potently enhanced antibody responses against coimmunized protein antigens in the absence of other exogenously added adjuvants. Elicitation of antibody responses against both virus-encoded antigens and coimmunized protein antigens was independent of the signaling via Toll-like receptors (TLRs) previously implicated in antiviral responses. In contrast, the adjuvant effect of rSFV on coimmunized protein was completely abolished in mice lacking the alpha/beta interferon (IFN-α/β) receptor (IFN-AR1), demonstrating that IFN-α/β signaling was critical for mediating this effect. Antibody responses directed against virus-encoded antigens were intact in IFN-AR1 −/− mice, suggesting that other signals are sufficient to drive immune responses against virally encoded antigens. These data provide a basis for the adjuvant effect of rSFV and show that different signals are required to stimulate antibody responses to virally encoded antigens and to antigens administered as purified protein vaccines, together with viral particles.

The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

BLyS is a survival cytokine that regulates peripheral B cell numbers and transitional B cell throughput. In mice, exogenous BLyS administration yields elevated pre-immune B cell numbers, an increased proportional representation of transitional B cells, and shifts in repertoire composition (1–3). Consistent with these observations, BLyS pretreatment alters the quality of antibody responses to HIV gp140 in mice, yielding enhanced neutralizing responses (4). While it is assumed that BLyS plays analogous roles in other species, similar studies in nonhuman primates are lacking. Accordingly, we have assessed the effects of exogenous BLyS treatment in rhesus macaques. Here we treated juvenile rhesus macaques with 0.05 mg/kg recombinant human BLyS over ten days. Physical exams and blood cell counts indicate no adverse effects of treatment except a decrease in lymphocyte counts during the treatment periods. We find that the numbers and proportional representation of transitional B cells (CD20+ IgM+ CD10+) increase during and immediately following BLyS treatment, returning to pre-treatment levels within 20–30 days. There is a small increase in plasma anti-dsDNA antibody, and a significant increase in anti-recombinant human BLyS antibody indicating immunogenicity in this species. Together, these results indicate that BLyS plays similar roles in rodents and primates. They further suggest that deliberate manipulation of BLyS may be an effective approach in rhesus macaques for expanding B cell repertoire diversity and altering the quality of antibody responses.

Multivalent molecules with repetitive structures including bacterial capsular polysaccharides and viral capsids elicit antibody responses through B cell receptor (BCR) crosslinking in the absence of T cell help. We report that immunization with these T cell–independent type 2 (TI-2) antigens causes up-regulation of endogenous retrovirus (ERV) RNAs in antigen-specific mouse B cells. These RNAs are detected via a mitochondrial antiviral signaling protein (MAVS)–dependent RNA sensing pathway or reverse-transcribed and detected via the cGAS-cGAMP-STING pathway, triggering a second, sustained wave of signaling that promotes specific immunoglobulin M production. Deficiency of both MAVS and cGAS, or treatment of MAVS-deficient mice with reverse transcriptase inhibitors, dramatically inhibits TI-2 antibody responses. These findings suggest that ERV and two innate sensing pathways that detect them are integral components of the TI-2 B cell signaling apparatus.

Protein-based vaccines require adjuvants to achieve optimal responses. Toll-like receptor (TLR) 9 agonists were previously shown to improve responses to protein-based vaccines, such as the Hepatitis B virus vaccine formulated in alum. Here, we used CpG-C together with the clinically relevant saponin-based adjuvant AbISCO-100/Matrix-M (AbISCO), to assess if TLR9 co-stimulation would quantitatively or qualitatively modulate HIV-1 envelope glycoprotein (Env)-specific B and T cell responses in rhesus macaques. The macaques were inoculated with soluble Env trimers in AbISCO, with or without the addition of CpG-C, using an interval similar to the Hepatitis B virus vaccine. Following a comprehensive evaluation of antigen-specific responses in multiple immune compartments, we show that the Env-specific circulating IgG, memory B cells and plasma cells displayed similar kinetics and magnitude in the presence or absence of CpG-C and that there was no apparent difference between the two groups in the elicited HIV-1 neutralizing antibody titers or antigen-specific CD4+ T cell responses. Importantly, the control of SHIV viremia was significantly improved in animals from both Env-immunized groups relative to adjuvant alone controls, demonstrating the potential of AbISCO to act as a stand-alone adjuvant for Env-based vaccines.

B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93(+)IgM(+)CD5(+)) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93(+)IgM(+)CD5(-) cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS.

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01., Author Summary The development of an HIV-1 vaccine that stimulates the production of antibodies capable of neutralizing diverse circulating HIV-1 strains remains a global priority. Studies have shown that broadly neutralizing antibodies (bNAbs) isolated from HIV-1 infected individuals can protect against infection in non-human primates and, in some cases, reduce viremia after already established infection. An intriguing feature of one class of bNAbs directed against the primary receptor binding site of HIV-1, the CD4 binding site (CD4bs), is that they are encoded by the same heavy chain gene segment, which encodes critical contacts for this class of Ab. Here, we asked if HIV-1 Env vaccination activates B cells that encode the rhesus macaque orthologue of this gene segment and if so what the genetic and structural properties of such antibodies are. We isolated a set of monoclonal antibodies encoded by this gene segment and demonstrate that one such Ab, GE356, binds the receptor binding site in a manner that is distinctly different from the mode of interaction of CD4bs-directed bNAbs. These results provide a possible explanation for the lack of broadly neutralizing activity following vaccination, even when antibodies encoded by gene segments frequently used in bNAbs are elicited.

HIV-1 neutralization requires Ab accessibility to the functional envelope glycoprotein (Env) spike. We recently reported the isolation of previously unidentified vaccine-elicited, CD4 binding site (CD4bs)-directed mAbs from rhesus macaques immunized with soluble Env trimers, indicating that this region is immunogenic in the context of subunit vaccination. To elucidate the interaction of the trimer-elicited mAbs with gp120 and their insufficient interaction with the HIV-1 primary isolate spike, we crystallized the Fab fragments of two mAbs, GE136 and GE148. Alanine scanning of their complementarity-determining regions, coupled with epitope scanning of their epitopes on gp120, revealed putative contact residues at the Ab/gp120 interface. Docking of the GE136 and GE148 Fabs to gp120, coupled with EM reconstructions of these nonbroadly neutralizing mAbs (non-bNAbs) binding to gp120 monomers and EM modeling to well-ordered trimers, suggested Ab approach to the CD4bs by a vertical angle of access relative to the more lateral mode of interaction used by the CD4bs-directed bNAbs VRC01 and PGV04. Fitting the structures into the available cryo-EM native spike density indicated clashes between these two vaccine-elicited mAbs and the topside variable region spike cap, whereas the bNAbs duck under this quaternary shield to access the CD4bs effectively on primary HIV isolates. These results provide a structural basis for the limited neutralizing breadth observed by current vaccine-induced, CD4bs-directed Abs and highlight the need for better ordered trimer immunogens. The analysis presented here therefore provides valuable information to guide HIV-1 vaccine immunogen redesign.

Signalling through Toll-like receptors (TLRs) by endogenous components of viruses or bacteria can promote antibody (Ab) isotype switching to IgG2a/c. Multiple cell types are capable of responding to TLR stimulation in vivo and the processes underlying TLR-induced Ab isotype switching are not fully defined. Here, we used feeble mice, which are deficient in the peptide/histidine transporter solute carrier family 15 member 4 (Slc15a4), and fail to produce cytokines including interferon alpha (IFNα) in response to TLR9 stimulation, to study Ab isotype switching to IgG2c in response to vaccination. We demonstrate that the production of IgG2c in response to CpGA-adjuvanted vaccines was severely reduced in feeble mice, while a more subtle defect was observed for CpGB. The reduced IgG2c production in feeble could not be ascribed to defective plasmacytoid dendritic cell (pDC) responses alone as we found that splenic cDCs and B cells from feeble mice were also defective in response to TLR9 ligation ex vivo. We conclude that Slc15a4 is required for intact function of TLR9-expressing cells and for effective Ab isotype switching to IgG2c in response to CpG-adjuvanted vaccines.

Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies.

Covalent conjugation of immune-stimulatory compounds to protein antigens is a potential means to self-adjuvant non-replicating subunit vaccines. Previously, it was demonstrated that covalent coupling of a Toll-like receptor (TLR) ligand to the exterior HIV-1 envelope glycoprotein, gp120, enhanced its immunogenicity. However, the consequences of chemical conjugation to gp120 on broadly neutralizing antibody (bNAb) epitopes were so far not examined. Here, we conjugated a TLR7/8 ligand to lysine residues on gp120 using NHS-PEO8-maleimide linkers and investigated if this affected Ab recognition of the CD4 binding site (CD4bs), a highly conserved target for bNAbs. We demonstrate that the recognition of the CD4bs was reduced following coupling, especially at a higher coupling ratio. These results have implications for the coupling of ligands to vaccine antigens where elicitation of humoral immune responses to specific neutralizing determinants is desired.

Semliki Forest virus nsP3 sequesters G3BP to inhibit stress granule formation on viral mRNAs. Furthermore, the efficient translation of viral mRNAs containing a translation enhancer element assists disruption of SGs in infected cells. This work thus describes a novel mechanism for SG disruption., Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.

The high overall genetic homology between human and rhesus macaques, coupled with the phenotypic conservation of lymphocyte populations, highlights the potential utility of non-human primates (NHPs) for the preclinical evaluation of vaccine candidates. For HIV-1, experimental models are needed to identify vaccine regimens capable of eliciting desired immune responses, such as broadly neutralizing antibodies. One important neutralization target on the HIV-1 envelope glycoproteins (Env) is the conserved primary CD4 receptor binding site (CD4bs). The isolation and characterization of CD4bs-specific neutralizing monoclonal antibodies (MAbs) from HIV-1 infected individuals has provided insights into how broadly reactive antibodies target this conserved epitope. In contrast, and for reasons that are not understood, current Env immunogens elicit CD4bs-directed antibodies with limited neutralization breadth. To facilitate the use of the NHP model to address this and other questions relevant to human humoral immunity, we defined features of the rhesus macaque immunoglobulin (Ig) loci and compared these to the human Ig loci. We then studied Env immunized rhesus macaques, identified single B-cells expressing CD4bs-specific antibodies, and sequenced and expressed a panel of functional MAbs. Comparison of vaccine-elicited MAbs with HIV-1 infection-induced MAbs revealed differences in the degree of somatic hypermutation of the Abs, as well as in the fine specificities targeted within the CD4bs. These data support the use of the preclinical NHP model to characterize vaccine-induced B cell responses at high resolution.

Using chemical germ-line mutagenesis, we screened mice for defects in the humoral immune response to a type II T-independent immunogen and an experimental alphavirus vector. A total of 26 mutations that impair humoral immunity were recovered, and 19 of these mutations have been positionally cloned. Among the phenovariants were bumble , cellophane , and Worker ascribed to mutations in Nfkbid , Zeb1 , and Ruvbl2 , respectively. We show that IκBNS, the nuclear IκB-like protein encoded by Nfkbid , is required for the development of marginal zone and peritoneal B-1 B cells and additionally required for extrafollicular antibody responses to T-independent and -dependent immunogens. Zeb1 is also required for marginal zone and peritoneal B-1 B-cell development as well as T-cell development, germinal center formation, and memory B-cell responses. Finally, Ruvbl2 is required for T-cell development and maximal T-dependent antibody responses. Collectively, the mutations that we identified give us insight into the points at which disruption of an antibody response can occur. All of the mutations identified to date directly affect lymphocyte development or function; none have an exclusive effect on cells of the innate immune system.

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.

The development and quality of a humoral immune response are largely influenced by the environment that supports the activation of naïve B cells. Human PDCs, through their unique capacity to produce high levels of IFN-α, have been shown earlier to enhance B cell responses stimulated by selected TLR ligands. In this study, we investigated whether PDCs also promote B cell activation induced by Th cell interactions and BCR ligation. Sorted human naive CD19+ CD27– B cells were activated in vitro with anti-Ig and irradiated CD4+ T cells. Under these conditions, the presence of supernatants from TLR-stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27high CD38high cells, and secretion of IgM. Similar results were observed when the B cells were activated in the presence of purified IFN-α. In contrast, supernatants from stimulated MDCs did not augment these functions. Also, IFN-α treatment of B cells up-regulated the expression of costimulatory molecule CD86 but not CD40, CD80, MHC class II, or CD25. Although direct IFN-α exposure of T cells suppressed their proliferative capacity, IFN-α treatment of B cells led to a small increase in their capacity to induce superantigen-driven activation of autologous CD4+ T cells. In summary, PDCs, via their production of IFN-α, may render B cells more responsive to T cell contact, which in turn, facilitates B cell proliferation and differentiation to antibody-producing cells.

The high-affinityin vivointeraction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact ofin vivoEnv-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by thein vivointeraction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.

The coxsackievirus–adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5–Lf complexes and this was dependent on high-mannose-typeN-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.

Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4 + T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein., Author Summary A major goal of HIV-1 vaccine research is to design novel candidates capable of neutralizing the vast array of viruses circulating in the human population. One approach is to base the vaccine upon the HIV-1 outer surface envelope glycoproteins to generate antibodies. However, during persistent infection in humans, the HIV-1 envelope glycoproteins have evolved structural features that limit the elicitation of broadly neutralizing antibodies. These immune “decoys” divert the antibody response resulting in virus subpopulations that can escape the host response. A potential means by which the virus elicits these decoy responses comes as a by-product of the entry process. Binding of the HIV-1 envelope glycoproteins to the primary receptor, human CD4, induces the formation of a second co-receptor binding site on the envelope glycoproteins, which then binds to another protein required for viral entry. Antibodies to the co-receptor binding site are generally ineffective at neutralizing HIV-1 patient isolates. Here, we demonstrate the mechanism by which antibodies to the HIV-1 co-receptor binding site are elicited in animals and humans injected with HIV-1 envelope glycoproteins and describe the implications of their formation regarding natural HIV-1 infection and vaccine design.

Viral vectors encoding heterologous vaccine antigens are potent inducers of cellular immune responses, but they are generally less efficient at stimulating humoral immunity. To improve the induction of antibody responses by Semliki Forest virus-based vaccines, a vector encoding a translation-enhancer element and a novel internal signal sequence for increased expression and secretion of soluble antigens was designed. Approximately tenfold more human immunodeficiency virus type 1 gp120 was secreted into culture supernatants of infected cells using the enhanced vector compared with the parental vector. This translated into a significant increase in gp120-specific antibodies in immunized mice, suggesting that antigen-expression levels from the parental vector are limiting for induction of antibody responses. These data encourage the use of the enhanced vector for elicitation of immune responses against heterologous antigens during vaccination.

Bimal K. Chakrabarti, Yu Feng, Shailendra Kumar Sharma, Krisha McKee, Gunilla B. Karlsson Hedestam, Celia C. LaBranche, David C. Montefiori, John R. Mascola, Richard T. Wyatt IAVI Neutralizing Antibody Center at The Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla, California, USA; The Vaccine Research Center, NIH, Bethesda, Maryland, USA; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA; The Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA

The field of DNA vaccines continues to advance and several new strategies to augment the immunogenicity of DNA vaccines are under evaluation. The majority of these studies are in the early preclinical stage, but some DNA vaccines have moved into clinical trials. In this review, we describe some of the more recent efforts aimed at increasing the immunogenicity of DNA vaccines, including the use of genetic adjuvants and plasmid-based expression of viral replicons. In addition, we discuss the possibility of using DNA vaccines to address emerging infectious agents where they may provide an advantage over other vaccine strategies and we review some areas where DNA vaccines have been used to target self-antigens.

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-αβ and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12°) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12° animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12° mice. A higher proportion of pDCs was activated in infected DAP12° mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-α and IFN-β. The homeostasis of WT and DAP12° pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12° bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-αβ production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-αβ production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.

Alpha/beta interferons (IFN-α/β) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-α/β are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-α/β production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-α/β in mDCs while fusion-defective virus did not induce IFN-α/β. The response to replication-defective virus was largely intact in MyD88 −/− mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-α/β in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-α/β in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

Vaccines based on recombinant viruses represent a promising strategy for the development of a prophylactic vaccine against HIV-1. However, despite a proven capacity to stimulate potent HIV-1-specific immune responses, viral systems have limited utility in homologous prime-boost regimens due to the generation of anti-vector immune responses. It is therefore important to develop a diverse set of vaccine candidates that can be combined in different heterologous prime-boost regimens and/or to identify a vaccine candidate that is less sensitive to anti-vector mediated immunity. In this report, we describe the design and pre-clinical immunogenicity of a Semliki Forest virus-based vaccine, VREP-C, encoding Indian origin HIV-1 clade C antigens. We show that a single immunization with VREP-C stimulates HIV-1-specific IFNgamma ELISPOT responses, which were efficiently boosted by a second and a third homologous VREP-C immunization resulting in highly potent cytotoxic T cell responses. These results suggest that VREP-C may be a valuable component of a future prophylactic vaccine against HIV-1.

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime∶boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime∶boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime∶boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime∶boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69