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The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ('supersite') on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRASeq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV23434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability., Introduction During the COVID-19 pandemic, more than 150 vaccine candidates were developed, but only a few have been licensed. Most licensed vaccines encode the full-length spike (S) protein, including 2 [...]

Author(s): Gopal Sapparapu [1, 2]; Estefania Fernandez [3]; Nurgun Kose [2]; Bin Cao [4]; Julie M. Fox [5]; Robin G. Bombardi [2]; Haiyan Zhao [3]; Christopher A. Nelson [3]; Aubrey [...]

Byline: Jessi Wildeson Jones (Biologics and Biomolecular Sciences, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut); Tiffani A. Greene (Integral Molecular, Inc., Philadelphia, Pennsylvania); Christine A. Grygon (Biologics and Biomolecular Sciences, Boehringer [...]

Byline: Jessi Wildeson Jones (Biologics and Biomolecular Sciences, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut); Tiffani A. Greene (Integral Molecular, Inc., Philadelphia, Pennsylvania); Christine A. Grygon (Biologics and Biomolecular Sciences, Boehringer [...]

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains in vitro and in vivo. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood-brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell-cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood-brain barrier disruption and vital entry into the central nervous system.

Certain chemokine receptors serve as cofactors for HIV type 1 envelope (env)-mediated cell-cell fusion and virus infection of CD4-positive cells. Macrophage tropic (M-tropic) HIV-1 isolates use CCR5, and T cell tropic (T-tropic) strains use CXCR4. To investigate the cofactors used by simian immunodeficiency viruses (SIV), we tested four T-tropic and two M-tropic SIV env proteins for their ability to mediate cell-cell fusion with cells expressing CD4 and either human or nonhuman primate chemokine receptors. Unlike HIV-1, both M- and T-tropic SIV envs used CCR5 but not CXCR4 or the other chemokine receptors tested. However, by testing a panel of CCR5/CCR2b chimeras, we found that the structural requirements for CCR5 utilization by M-tropic and T-tropic SIV strains were different. T-tropic SIV strains required the second extracellular loop of CCR5 whereas a closely related M-tropic SIV strain could, like M-tropic HIV-1 strains, use the amino-terminal domain of CCR5. As few as two amino acid changes in the SIV env V3 domain affected the regions of CCR5 that were critical for fusogenic activity. Receptor signaling was not required for either fusion or infection. Our results suggest that vital tropism may be influenced not only by the coreceptors used by a given virus strain but also by how a given coreceptor is used.

The beta-chemokine receptors CCR5 and CCR2b are the cofactors for entry of M-tropic viruses into cells. Previous studies have reported that M-tropic trains are more sensitive to substitutions in the regions of CCR5 than CCR2b. To understand the molecular basis of CCR5 usage in viral tropism, chimeric molecules of both CCR5 and CCR2b were constructed and regions involved in cofactor utilization by HIV-1 strains were mapped. The chimeras showed that the amino terminus and the first extracellular loop of CCR5 contribute to enhancing the entry of HIV-1 cofactors into the cell.

A mutant allele of the CC-chemokine receptor-5, which is common in caucasians is involved in resistance to HIV-1 infection. None of the HIV-1 infected caucasians in a cohort are homozygous for the mutation, while the frequency of heterozygotes is 35% less than that in normal populations. The white blood cells in homozygous individuals with null alleles are highly resistant to the HIV-1 virus, while the lower frequency of heterozygotes implied partial resistance in seropositive Caucasians. The mutation prevents HIV infection by inhibiting membrane fusion.

HIV-1 isolate 89.6 uses Fusin and CKR-5 as entry cofactors indicating that the evolution from a M-tropic to T-tropic phenotype probably occurs due to changes in HIV-1 env that allow the use of dual cofactors. The beta-chemokine receptors CKR-3 and CHR-2b allow the formation of syncytia mediated by HIV-1 89.6 env proteins but fail to allow fusion by T-tropic and M-tropic strains. Expression of CKR-5 with CD4 allows nonpermissive cells to form syncytia with cells expressing M-tropic proteins. However, CKR-5 fails to allow entry of cells expressing T-tropic and HIV-1 env proteins.

Retroviral vectors containing CD4 and an appropriate chemokine receptor were evaluated for the ability to transduce cells infected with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). These CD4-chemokine receptor pseudotypes were able to target HIV- and Sly-infected cell lines and monocyte-derived macrophages in a manner that corresponded to the specificity of the viral envelope glycoprotein for its CD4-chemokine receptor complex. This approach could offer a way to deliver antiviral genes directly to HIV-infected cells in vivo and could provide an additional treatment strategy in conjunction with existing antiviral therapies., Treatment of HIV-infected patients with combinations of antiretroviral drugs has resulted in a profound reduction of detectable virus in plasma and lymph tissue and is expected to have considerable clinical [...]

A high-throughput approach termed shotgun mutagenesis is used for mapping the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids comprising the critical contact points of each epitope are identified, which has defined the immonodominant structures of the GPCR and their physicochemistry.

Virus-like particles carrying conformationally complex membrane proteins are used as soluble probes of membrane protein interactions. These particles have served as versatile probes for screening crude and purified antibody preparations for receptor specificity, epitope reactivity, and relative binding kinetics.