Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

A critical roadblock to HIV vaccine development is the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans. In people living with HIV-1, bnAbs take years to develop. The HVTN 133 clinical trial studied a peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs (NCT03934541). Here, we report MPER peptide-liposome induction of polyclonal HIV-1 B cell lineages of mature bnAbs and their precursors, the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains with lineage initiation after the second immunization. Neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids. This study demonstrates proof of concept for rapid vaccine induction of human B cell lineages with heterologous neutralizing activity and selection of antibody improbable mutations and outlines a path for successful HIV-1 vaccine development.

In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies.

SARS-CoV-2-neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) or the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo, increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

RAB11FIP5 Expression and Altered Natural Killer Cell Function Are Associated with Induction of HIV Broadly Neutralizing Antibody Responses.

HIV-1 broadly neutralizing antibodies (bnAbs) are difficult to induce with vaccines but are generated in ∼50% of HIV-1-infected individuals. Understanding the molecular mechanisms of host control of bnAb induction is critical to vaccine design. Here, we performed a transcriptome analysis of blood mononuclear cells from 47 HIV-1-infected individuals who made bnAbs and 46 HIV-1-infected individuals who did not and identified in bnAb individuals upregulation of RAB11FIP5, encoding a Rab effector protein associated with recycling endosomes. Natural killer (NK) cells had the highest differential expression of RAB11FIP5, which was associated with greater dysregulation of NK cell subsets in bnAb subjects. NK cells from bnAb individuals had a more adaptive/dysfunctional phenotype and exhibited impaired degranulation and cytokine production that correlated with RAB11FIP5 transcript levels. Moreover, RAB11FIP5 overexpression modulated the function of NK cells. These data suggest that NK cells and Rab11 recycling endosomal transport are involved in regulation of HIV-1 bnAb development.

International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models.

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1-infected individuals, bnAbs evolved from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar antibody maturation process. One way to test a vaccine is to immunize mouse models that express human bnAb precursors and assess whether the vaccine can convert precursor antibodies into bnAbs. A major problem with such mouse models is that bnAb expression often hinders B cell development. Such developmental blocks may be attributed to the unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are usually under negative selection during primary B cell development. To address this problem, we devised a method to circumvent such B cell developmental blocks by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of the VRC26UCA led to developmental arrest of B cell progenitors in bone marrow; poly-reactivity of the VRC26UCA and poor pairing of the VRC26UCA heavy chain with the mouse surrogate light chain may contribute to this phenotype. The conditional expression strategy bypassed the impediment to VRC26UCA B cell development, enabling the expression of VRC26UCA in mature B cells. This approach should be generally applicable for expressing other bnAbs that are under negative selection during B cell development.

Murine Glucocorticoid Receptors Orchestrate B Cell Migration Selectively between Bone Marrow and Blood.

Glucocorticoids promote CXCR4 expression by T cells, monocytes, macrophages, and eosinophils, but it is not known if glucocorticoids regulate CXCR4 in B cells. Considering the important contributions of CXCR4 to B cell development and function, we investigated the glucocorticoid/CXCR4 axis in mice. We demonstrate that glucocorticoids upregulate CXCR4 mRNA and protein in mouse B cells. Using a novel strain of mice lacking glucocorticoid receptors (GRs) specifically in B cells, we show that reduced CXCR4 expression associated with GR deficiency results in impaired homing of mature B cells to bone marrow, whereas migration to other lymphoid tissues is independent of B cell GRs. The exchange of mature B cells between blood and bone marrow is sensitive to small, physiologic changes in glucocorticoid activity, as evidenced by the lack of circadian rhythmicity in GR-deficient B cell counts normally associated with diurnal patterns of glucocorticoid secretion. B cellGRKO mice mounted normal humoral responses to immunizations with T-dependent and T-independent (Type 1) Ags, but Ab responses to a multivalent T-independent (Type 2) Ag were impaired, a surprise finding considering the immunosuppressive properties commonly attributed to glucocorticoids. We propose that endogenous glucocorticoids regulate a dynamic mode of B cell migration specialized for rapid exchange between bone marrow and blood, perhaps as a means to optimize humoral immunity during diurnal periods of activity.

Simian-Human Immunodeficiency Virus SHIV.CH505-Infected Infant and Adult Rhesus Macaques Exhibit Similar Env-Specific Antibody Kinetics, despite Distinct T-Follicular Helper and Germinal Center B Cell Landscapes.

Global elimination of pediatric human immunodeficiency virus (HIV) infections will require the development of novel immune-based approaches, and understanding infant immunity to HIV is critical to guide the rational design of these intervention strategies. Despite their immunological immaturity, chronically HIV-infected children develop broadly neutralizing antibodies (bnAbs) more frequently and earlier than adults do. However, the ontogeny of humoral responses during acute HIV infection is poorly defined in infants and challenging to study in human cohorts due to the presence of maternal antibodies. To further our understanding of age-related differences in the development of HIV-specific immunity during acute infection, we evaluated the generation of virus-specific humoral immune responses in infant (n = 6) and adult (n = 12) rhesus macaques (RMs) infected with a transmitted/founder (T/F) simian-human immunodeficiency virus (SHIV) (SHIV.C.CH505 [CH505]). The plasma HIV envelope-specific IgG antibody kinetics were similar in SHIV-infected infant and adult RMs, with no significant differences in the magnitude or breadth of these responses. Interestingly, autologous tier 2 virus neutralization responses also developed with similar frequencies and kinetics in infant and adult RMs, despite infants exhibiting significantly higher follicular T helper cell (Tfh) and germinal center B cell frequencies than adults. Finally, we show that plasma viral load was the strongest predictor of the development of autologous virus neutralization in both age groups. Our results indicate that the humoral immune response to SHIV infection develops with similar kinetics among infant and adult RMs, suggesting that the early-life immune system is equipped to respond to HIV-1 and promote the production of neutralizing HIV antibodies.IMPORTANCE There is a lack of understanding of how the maturation of the infant immune system influences immunity to HIV infection or how these responses differ from those of adults. Improving our knowledge of infant HIV immunity will help guide antiviral intervention strategies that take advantage of the unique infant immune environment to successfully elicit protective immune responses. We utilized a rhesus macaque model of SHIV infection as a tool to distinguish the differences in HIV humoral immunity in infants versus adults. Here, we demonstrate that the kinetics and quality of the infant humoral immune response to HIV are highly comparable to those of adults during the early phase of infection, despite distinct differences in their Tfh responses, indicating that slightly different mechanisms may drive infant and adult humoral immunity.

The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center.

T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

Germinal Center Hypoxia Potentiates Immunoglobulin Class Switch Recombination.

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.

Identification of a tissue-specific, C/EBPß-dependent pathway of differentiation for murine peritoneal macrophages.

Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-ß. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBPß-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBPß plays an intrinsic, tissue-restricted role in the generation of resident macrophages.

Immunogenicity of membrane-bound HIV-1 gp41 membrane-proximal external region (MPER) segments is dominated by residue accessibility and modulated by stereochemistry.

Structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity. By contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed membrane-proximal external region (MPER) of HIV-1 glycoprotein 41 as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while preserving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent-exposed MPER tryptophan residue (Trp-680) was immunodominant, focusing immune responses, despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for immunogen design aimed at targeting B cell antibody responses.

Activation-induced cytidine deaminase mediates central tolerance in B cells.

The Aicda gene product, activation-induced cytidine deaminase (AID), initiates somatic hypermutation, class-switch recombination, and gene conversion of Ig genes by the deamination of deoxycytidine, followed by error-prone mismatch- or base-excision DNA repair. These processes are crucial for the generation of genetically diverse, high affinity antibody and robust humoral immunity, but exact significant genetic damage and promote cell death. In mice, physiologically significant AID expression was thought to be restricted to antigen-activated, mature B cells in germinal centers. We now demonstrate that low levels of AID in bone marrow immature and transitional B cells suppress the development of autoreactivity. Aicda(-/-) mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature and transitional B cells. In vitro, AID deficient immature/transitional B cells are significantly more resistant to anti-IgM-induced apoptosis than their normal counterparts. Thus, early AID expression plays a fundamental and unanticipated role in purging self-reactive immature and transitional B cells during their maturation in the bone marrow.

A HIV-1 Gp41 Peptide-Liposome Vaccine Elicits Neutralizing Epitope-Targeted Antibody Responses in Healthy Individuals.

Background: HIV-1 vaccine development is a global health priority. Broadly neutralizing antibodies (bnAbs) which target the HIV-1 gp41 membrane-proximal external region (MPER) have some of the highest neutralization breadth. An MPER peptide-liposome vaccine has been found to expand bnAb precursors in monkeys. Methods: The HVTN133 phase 1 clinical trial (NCT03934541) studied the MPER-peptide liposome immunogen in 24 HIV-1 seronegative individuals. Participants were recruited between 15 July 2019 and 18 October 2019 and were randomized in a dose-escalation design to either 500 mcg or 2000 mcg of the MPER-peptide liposome or placebo. Four intramuscular injections were planned at months 0, 2, 6, and 12. Results: The trial was stopped prematurely due to an anaphylaxis reaction in one participant ultimately attributed to vaccine-associated polyethylene glycol. The immunogen induced robust immune responses, including MPER+ serum and blood CD4+ T-cell responses in 95% and 100% of vaccinees, respectively, and 35% (7/20) of vaccine recipients had blood IgG memory B cells with MPER-bnAb binding phenotype. Affinity purification of plasma MPER+ IgG demonstrated tier 2 HIV-1 neutralizing activity in two of five participants after 3 immunizations. Conclusions: MPER-peptide liposomes induced gp41 serum neutralizing epitope-targeted antibodies and memory B-cell responses in humans despite the early termination of the study. These results suggest that the MPER region is a promising target for a candidate HIV vaccine.

Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

In utero human cytomegalovirus infection expands NK cell-like FcγRIII-expressing CD8+ T cells that mediate antibody-dependent functions.

Human cytomegalovirus (HCMV) profoundly modulates host T and natural killer (NK) cells across the lifespan, expanding unique effector cells bridging innate and adaptive immunity. Though HCMV is the most common congenital infection worldwide, how this ubiquitous herpesvirus impacts developing fetal T and NK cells remains unclear. Using computational flow cytometry and transcriptome profiling of cord blood from neonates with and without congenital HCMV (cCMV) infection, we identify major shifts in fetal cellular immunity marked by an expansion of Fcγ receptor III (FcγRIII)-expressing CD8+ T cells (FcRT) following HCMV exposure in utero. FcRT cells from cCMV-infected neonates express a cytotoxic NK cell-like transcriptome and mediate antigen-specific antibody-dependent functions including degranulation and IFNγ production, the hallmarks of NK cell antibody-dependent cellular cytotoxicity (ADCC). FcRT cells may represent a previously unappreciated effector population with innate-like functions that could be harnessed for maternal-infant vaccination strategies and antibody-based therapeutics in early life.

Conjugation of HIV-1 envelope to hepatitis B surface antigen alters vaccine responses in rhesus macaques.

An effective HIV-1 vaccine remains a critical unmet need for ending the AIDS epidemic. Vaccine trials conducted to date have suggested the need to increase the durability and functionality of vaccine-elicited antibodies to improve efficacy. We hypothesized that a conjugate vaccine based on the learned response to immunization with hepatitis B virus could be utilized to expand T cell help and improve antibody production against HIV-1. To test this, we developed an innovative conjugate vaccine regimen that used a modified vaccinia virus Ankara (MVA) co-expressing HIV-1 envelope (Env) and the hepatitis B virus surface antigen (HBsAg) as a prime, followed by two Env-HBsAg conjugate protein boosts. We compared the immunogenicity of this conjugate regimen to matched HIV-1 Env-only vaccines in two groups of 5 juvenile rhesus macaques previously immunized with hepatitis B vaccines in infancy. We found expansion of both HIV-1 and HBsAg-specific circulating T follicular helper cells and elevated serum levels of CXCL13, a marker for germinal center activity, after boosting with HBsAg-Env conjugate antigens in comparison to Env alone. The conjugate vaccine elicited higher levels of antibodies binding to select HIV Env antigens, but we did not observe significant improvement in antibody functionality, durability, maturation, or B cell clonal expansion. These data suggests that conjugate vaccination can engage both HIV-1 Env and HBsAg specific T cell help and modify antibody responses at early time points, but more research is needed to understand how to leverage this strategy to improve the durability and efficacy of next-generation HIV vaccines.

Neonatal SHIV infection in rhesus macaques elicited heterologous HIV-1-neutralizing antibodies.

Infants and children infected with human immunodeficiency virus (HIV)-1 have been shown to develop neutralizing antibodies (nAbs) against heterologous HIV-1 strains, characteristic of broadly nAbs (bnAbs). Thus, having a neonatal model for the induction of heterologous HIV-1 nAbs may provide insights into the mechanisms of neonatal bnAb development. Here, we describe a neonatal model for heterologous HIV-1 nAb induction in pathogenic simian-HIV (SHIV)-infected rhesus macaques (RMs). Viral envelope (env) evolution showed mutations at multiple sites, including nAb epitopes. All 13 RMs generated plasma autologous HIV-1 nAbs. However, 8/13 (62%) RMs generated heterologous HIV-1 nAbs with increasing potency over time, albeit with limited breadth, and mapped to multiple nAb epitopes, suggestive of a polyclonal response. Moreover, plasma heterologous HIV-1 nAb development was associated with antigen-specific, lymph-node-derived germinal center activity. We define a neonatal model for heterologous HIV-1 nAb induction that may inform future pediatric HIV-1 vaccines for bnAb induction in infants and children.

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies.

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and, in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. To precisely engineer bnAb boosting immunogens, we used molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identified Env mutations that were predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encoded antibody affinity gains and selected for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.