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Hangartner, L.

Publications and source records attributed to Hangartner, L..

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Germinal centers can persist for up to one year with declining somatic hypermutation and ongoing plasma cell output

The total duration of primary germinal center (GC) responses, and how B cell evolution changes as these responses mature, remain poorly defined. Here, longitudinal sampling of rhesus macaques immunized with HIV Envelope protein revealed that a single slow-delivery prime sustained ongoing GCs for nearly a year without additional antigen. Somatic hypermutation (SHM) accumulated throughout these prolonged responses, but its rate declined substantially over time, independent of both priming strategy and boosting. Over the same period, subdominant non-base Env specificities became increasingly represented within the GC response. Delaying boosting until after primary GC contraction increased recruitment of recalled memory B cells into the GC response. Bone marrow plasma cells were generated throughout prolonged GC maturation, with the timing of their generation modulated by booster timing. Together, these findings reveal how GCs evolve across nearly a year and how this prolonged maturation shapes the memory and plasma cell compartments that underpin long-term humoral immunity.

immunology↗

Structure and functional diversity of antibodies targeting the P. falciparum circumsporozoite protein C-terminal domain

The Plasmodium falciparum circumsporozoite protein (PfCSP) is the major surface antigen on Pf sporozoites. WHO-recommended vaccines RTS,S/AS01E and R21/Matrix-M target the PfCSP major repeat region and C-terminal domain (ctCSP). Although multiple studies associated protection with antibody responses to ctCSP, only a few ctCSP-specific monoclonal antibodies (mAbs) have been characterized. Here, crystal structures of 11 Fab-ctCSP complexes reveal how mAbs against the conserved {beta}-epitope region achieve diverse modes of strain-transcending recognition, in contrast to mAbs to the hypervariable -epitope. Consistent with previous studies, ctCSP on sporozoites could be unmasked by mAbs that bind CSP repeats, with unmasking dependent on the mAb fine-specificity and binding mode. In vitro, ctCSP mAbs promoted stronger Fc-receptor signaling, cellular cytotoxicity, and phagocytosis than repeat region mAbs, while mAb combinations targeting distinct PfCSP epitopes modulated Fc-signaling and cellular cytotoxicity. This study provides a rationale for optimization of PfCSP-based immunogens to enhance Fc-mediated contributions to malaria vaccine efficacy.

microbiology↗

Modulation of antigen delivery and lymph node activation in non-human primates by saponin adjuvant SMNP

Saponin-based vaccine adjuvants are potent in preclinical animal models and humans, but their mechanisms of action remain poorly understood. Here, using a stabilized HIV envelope trimer immunogen, we carried out studies in non-human primates (NHPs) comparing the most common clinical adjuvant alum with Saponin/MPLA Nanoparticles (SMNP), a novel ISCOMs-like adjuvant. SMNP elicited substantially stronger humoral immune responses than alum, including 7-fold higher peak antigen-specific germinal center B cell responses, 18-fold higher autologous neutralizing antibody titers, and higher levels of antigen-specific plasma and memory B cells. PET-CT imaging in live NHPs showed that, unlike alum, SMNP promoted rapid antigen accumulation in both proximal and distal lymph nodes (LNs). SMNP also induced strong type I interferon transcriptional signatures, expansion of innate immune cells, and increased antigen presenting cell activation in LNs. These findings indicate that SMNP promotes multiple facets of the early immune response relevant for enhanced immunity to vaccination.

bioengineering↗

Dose-dependent regulation of immune memory responses against HIV by saponin monophosphoryl lipid A nanoparticle adjuvant

The induction of durable protective immune responses is the main goal of prophylactic vaccines, and adjuvants play an important role as drivers of such responses. Despite advances in vaccine strategies, a safe and effective HIV vaccine remains a significant challenge. The use of an appropriate adjuvant is crucial to the success of HIV vaccines. Here we assessed the saponin/MPLA nanoparticle (SMNP) adjuvant with an HIV envelope (Env) trimer, evaluating the safety and impact of multiple variables including adjuvant dose (16-fold dose range), immunization route, and adjuvant composition on the establishment of Env-specific memory T and B cell responses (TMem and BMem) and long-lived plasma cells in non-human primates. Robust BMem were detected in all groups, but a 6-fold increase was observed in the highest SMNP dose group vs. the lowest dose group. Similarly, stronger vaccine responses were induced in the highest SMNP dose for CD40L+OX40+ CD4 TMem (11-fold), IFN{gamma}+ CD4 TMem (15-fold), IL21+ CD4 TMem (9-fold), circulating TFH (3.6-fold), bone marrow plasma cells (7-fold), and binding IgG (1.3-fold). Substantial tier-2 neutralizing antibodies were only observed in the higher SMNP dose groups. These investigations highlight the dose-dependent potency of SMNP in non-human primates, which are relevant for human use and next-generation vaccines.

immunology↗

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for detectable viral neutralization. Author SummaryAntibodies that bind to the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) on virions can neutralize viral infectivity. Antibodies may also bind to Env on the surface of virus-infected cells and recruit immune cells to eliminate the productively infected cells through a process known as antibody dependent cellular cytotoxicity (ADCC). In rare instances, certain antibodies are capable of mediating ADCC despite negligible neutralizing activity. Such antibodies are thought to have contributed to the modest protection observed in the RV144 HIV-1 vaccine trial and in some nonhuman primate studies. One antibody, PGT145, was found to cross-react with simian immunodeficiency virus (SIV) and to mediate potent ADCC against SIV-infected cells despite weak neutralization of viral infectivity. We therefore tested if the potent ADCC activity of PGT145 could protect rhesus macaques against mucosal challenge with pathogenic SIV. PGT145 did not protect against wild-type SIVmac239, but did protect against an SIVmac239 variant with a single amino acid substitution in Env (K180S) that increases antibody binding to Env and makes the virus susceptible to neutralization. Thus, while ADCC may contribute to protection against immunodeficiency viruses through the elimination of productively infected cells, the higher affinity of Env binding necessary for potent neutralization is a critical determinant of antibody-mediated protection.

microbiology↗

Long-lasting germinal center responses to a priming immunization with continuous proliferation and somatic mutation

Germinal centers (GCs) are the engines of antibody evolution. Using HIV Env protein immunogen priming in rhesus monkeys (RM) followed by a long period without further immunization, we demonstrate GC B cells (BGC) lasted at least 6 months (29 weeks), all the while maintaining rapid proliferation. A 186-fold BGC cell increase was present by week 10 compared to a conventional immunization. Single cell transcriptional profiling revealed that both light zone and dark zone GC states were sustained throughout the 6 months. Antibody somatic hypermutation (SHM) of BGC cells continued to accumulate throughout the 29 week priming period, with evidence of selective pressure. Additionally, Env-binding BGC cells were still 49-fold above baseline 29 weeks after immunization, suggesting that they could be active for significantly longer periods of time. High titers of HIV neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing significant immunodominance challenges for B cells, among other difficulties. Memory B cells (BMem) generated under these long priming conditions had higher levels of SHM, and both BMem cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning the >6-month GC period were identified, demonstrating continuous GC activity and selection for at least 191 days, with no additional antigen exposure. A long prime, adjuvanted, slow delivery (12-day) immunization approach holds promise for difficult vaccine targets, and suggests that patience can have great value for tuning GCs to maximize antibody responses.

immunology↗

Mapping polyclonal antibody responses in non-human primates vaccinated with HIV Env trimer subunit vaccines

Rational immunogen design aims to focus antibody responses to vulnerable sites on the primary antigens. Given the size of these antigens there is however potential for eliciting unwanted, off-target responses. Here, we used our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized, and the approach angle by which these antibodies bound, constituted a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are responsible for some neutralization breadth. Moreover, cryoEM analysis of a fully-protected animal revealed a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials earlier this year.

immunology↗

Vaccine-induced protection from homologous Tier 2 simian-human immunodeficiency virus challenge in nonhuman primates

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (Tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection following vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers from the BG505 HIV isolate. Repeat intrarectal challenge with homologous Tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. In contrast, high-titer animals demonstrated protection that was gradually lost as nAb titers waned over weeks to months. From these results, we determined that an autologous serum ID50 nAb titer of ~1:500 was required to afford over 90% protection from medium-dose SHIV infection. We further identified autologous nAb titers, but not ADCC or T cell activity, as strong correlates of protection. These results provide proof-of-concept that Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing Tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

immunology↗

Reprogramming the antigen specificity of B cells using genome-editing technologies

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.

immunology↗