Biology subjects

Rakasz, E. G.

Publications and source records attributed to Rakasz, E. G..

7 recordsLinked to original sources

bioRxiv2025

Harnessing mRNA for the expression of monoclonal IgG and IgA in non-human primates.

Monoclonal antibodies (mAbs) are an increasingly essential class of medicines across many disease areas (1). In the human body, there are five antibody isotypes, each with potential prophylactic or therapeutic benefits for different disease indications. However, 97% of all clinically approved mAbs are produced as the IgG isotype, partly due to differences in half-life, but largely due to challenges associated with recombinantly producing non-IgG isotypes like IgM or IgA, which have additional N-linked glycan sites and can present as multivalent oligomers. One potential solution to this challenge is to express mAbs in situ using mRNA encapsulated in lipid nanoparticles (LNP), bypassing the need for recombinant protein production (2). Here, we demonstrate the feasibility of expressing a mAb as both IgG and IgA in non-human primates (NHPs) using mRNA-LNPs. We express ePGDM1400v9, a broadly neutralizing mAb targeting human immunodeficiency virus (HIV), in both IgG1 and IgA2 formats by infusing NHPs with LNPs containing the appropriate mRNA. Though IgG1 expression levels were higher than those of IgA2, both formats were detectable in serum within one day of LNP infusion in all NHPs, and both were detectable in mucosal secretions of most animals. Importantly, serum mRNA-produced IgG1 and IgA2 retained HIV-neutralizing function. Furthermore, mass spectrometry analysis confirmed that mAbs of either isotype produced in situ exhibited glycosylation patterns highly similar to that of native antibody, which is likely to confer therapeutic advantages. Altogether, this work demonstrates the feasibility of using mRNA-LNPs to express native-like mAbs of non-IgG isotypes in primates and enables further development of non-IgG mAb constructs. SignificanceMonoclonal antibodies (mAbs) are a rapidly growing class of essential medicines across diverse disease areas and applications. However, the impact potential of mAbs is limited by challenges in production and purification, which favors the use of the IgG antibody isotype despite the disease-specific advantages that other isotypes might offer. One way to overcome this challenge is to express mAbs in situ using messenger ribonucleic acid delivered by lipid nanoparticles (mRNA-LNPs). Here, we show that an anti-human immunodeficiency virus (HIV) mAb, ePGDM1400v9, can be expressed as two different antibody isotypes, IgG and IgA, in nonhuman primates (NHPs) by mRNA-LNP delivery. We demonstrate that both mAb isotypes retain function and exhibit native-like glycosylation patterns that are not achievable with conventional recombinant mAbs.

immunology↗

bioRxiv2021

Molecular insights into antibody-mediated protection against the prototypic simian immunodeficiency virus

SIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated low dose intraveneous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

immunology↗

bioRxiv2021

CAR/CXCR5-T cell immunotherapy is safe and potentially efficacious in promoting sustained remission of SIV infection

During chronic human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection prior to AIDS progression, the vast majority of viral replication is concentrated within B cell follicles of secondary lymphoid tissues. We investigated whether infusion of T cells expressing an SIV-specific chimeric antigen receptor (CAR) and the follicular homing receptor, CXCR5, could successfully kill viral-RNA+ cells in targeted lymphoid follicles in SIV-infected rhesus macaques. In this study, CD4 and CD8 T cells from rhesus macaques were genetically modified to express antiviral CAR and CXCR5 moieties (generating CAR/CXCR5-T cells) and autologously infused into a chronically infected animal. At 2 days post-treatment, the CAR/CXCR5-T cells were located primarily in spleen and lymph nodes both inside and outside of lymphoid follicles. Few CAR/CXCR5-T cells were detected in the rectum and lung, and no cells were detected in the bone marrow, liver, brain, or ileum. Within follicles, CAR/CXCR5-T cells were found in direct contact with SIV viral RNA+ cells. We next infused CAR/CXCR5-T cells into ART-suppressed SIV-infected rhesus macaques, in which the animals were released from ART at the time of infusion. These CAR/CXCR5-T cells replicated in vivo within both the extrafollicular and follicular regions of lymph nodes and accumulated within lymphoid follicles. CAR/CXR5-T cell concentrations in follicles peaked during the first week post-infusion but declined to undetectable levels after 2 to 4 weeks. Overall, CAR/CXCR5-T cell-treated animals maintained lower viral loads and follicular viral RNA levels than untreated control animals, and no outstanding adverse reactions were noted. These findings indicate that CAR/CXCR5-T cell treatment is safe and holds promise as a future treatment for the durable remission of HIV. Author summaryA person infected with human immunodeficiency virus (HIV) has replicating virus concentrated within the follicles of lymphoid tissues. The cells needed to clear the infection, cytotoxic T lymphocytes, have limited access to follicles and, thus, the cytotoxic T lymphocytes are never completely able to clear all of the HIV from the body. In this study, we have produced immunotherapeutic T cells that home to follicles and clear infected cells. These T cells express a viral targeting chimeric antigen receptor (CAR) and a molecule called CXCR5, which leads to homing of the cells to follicles. Upon administration of these CAR T-cells to virus-infected primates, we found that the cells localized to the follicle, replicated, and directly interacted with infected cells. While the cells were not maintained in the animals for more than 4 weeks, most of the treated animals maintained lower levels of virus in the blood and follicles than untreated control animals. This study shows that this immunotherapy has potential as a treatment leading to long-term remission of HIV without the need for antiretroviral drugs.

immunology↗

bioRxiv2021

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

immunology↗

bioRxiv2020

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

immunology↗

bioRxiv2018

Absence of SIV-specific CD8+ T cells in germinal centers may set the stage for persistent infection

CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection.\n\nAuthor SummaryA paucity of SIV-specific CD8+ T cells in lymphoid follicles and complete absence within most follicular germinal centers during early infection may set the stage for the establishment of persistent chronic infection.

immunology↗

bioRxiv2017

Highly efficient maternal-fetal Zika virus transmission in pregnant rhesus macaques

Infection with Zika virus (ZIKV) is associated with human congenital fetal anomalies. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While non-pregnant animals clear viremia within 10-12 days, maternal viremia was prolonged in 3 of 4 pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section. In all pregnancies, neutrophilic infiltration was present at the maternal-fetal interface (decidua, placenta, fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerve (first trimester infection only). Consistent vertical transmission in this primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection.

pathology↗