Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline–revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Alphaviruses are enveloped, positive sense single-stranded RNA viruses that can cause significant human diseases ranging from arthritis to encephalitis (1–3). Alphaviruses that are associated with musculoskeletal disease (arthritogenic alphaviruses) include Chikungunya virus (CHIKV), Mayaro virus (MAYV), Ross River virus (RRV), O’nyong-nyong virus (ONNV), and others; these viruses are globally distributed and transmitted by mosquitos. Symptomatic infection by arthritogenic alphaviruses is characterized by fever, rash, myalgia, as well as both acute and chronic peripheral polyarthralgia (4, 5). The arthropathy can be debilitating and persist for months to years after infection. More severe manifestations of alphavirus disease—including encephalopathy and mortality—have been reported (6, 7). These viruses cause endemic disease as well as large, sporadic global outbreaks (8–10). Currently, there are no approved vaccines or antiviral therapies for the prevention or treatment of alphavirus infection.MAYV is an arthritogenic alphavirus that was first isolated in 1954 in Trinidad, and recent outbreaks have been reported in numerous areas of Central and South America (11, 12). The primary vectors for MAYV are Haemagogus spp. mosquitoes, which transmit the virus to primates in a sylvatic cycle. However, MAYV vector competence studies have demonstrated transmission potential in multiple Aedes and Anopheles mosquitoes (13–17). The wide range and distribution of MAYV-competent vectors underscores the risk of potential urban transmission (18) and global spread (19).The alphavirus glycoprotein is composed of heterodimers of two transmembrane subunits, E2 and E1, which mediate viral attachment and membrane fusion, respectively (20–22). The prefusion E2/E1 heterodimer forms a trimeric spike that is arranged in an icosahedral lattice on the viral particle. E2 is initially expressed as a precursor polypeptide known as p62. During virus biogenesis, p62 is processed by cellular furin to generate E2 and the peripheral E3 polypeptide. E3 remains bound to the E2/E1 heterodimer during exocytic transport and prevents premature conformational changes and membrane fusion (23, 24). The release of E3 is the final step of virus maturation and primes the glycoprotein for membrane fusion.Both E2 and E1 proteins are targets of the neutralizing antibody response. Antibody-mediated protection by neutralizing monoclonal antibodies (mAbs) has been shown against several alphaviruses (25–31). We and others have reported the isolation of potent and protective neutralizing CHIKV mAbs targeting regions of E2, such as the β-connector region and the A domain (25–27). These mAbs neutralize viral particles via multiple mechanisms, including the prevention of attachment and membrane fusion. The alphavirus receptor Mxra8 binds to regions spanning the A and B domains of E2 protein (32), and neutralizing mAbs targeting these regions can effectively disrupt virus interaction with the host receptor (33).Many of the identified, neutralizing human mAbs against alphaviruses are virus-specific and do not inhibit heterologous alphaviruses. Notably, most of these mAbs target CHIKV, and there are few examples of MAYV-reactive human mAbs. Recent work has demonstrated the cross-reactivity and cross-neutralization of human polyclonal sera to heterologous alphaviruses (34–36), suggesting that broadly reactive and/or broadly neutralizing monoclonal antibodies (bNAbs) may be elicited by alphavirus infection in humans. While a number of murine bNAbs have been characterized (30, 37, 38), few human bNAbs that engage multiple alphaviruses have been described (33). For example, the murine mAb CHK-265 can protect against CHIKV, MAYV, and RRV challenge in mice (38). More recently, a human mAb, RRV-12, was shown to protect mice against RRV and MAYV infection (33). Both CHK-265 and RRV-12 broadly neutralize infection by engaging the B domain of E2, but whether such protective alphavirus bNAbs are elicited commonly during the course of human CHIKV infection is unknown.Here, we describe the isolation and characterization of cross-reactive alphavirus mAbs from a CHIKV-convalescent donor. We employed a single B cell sorting strategy using a heterologous MAYV antigen to isolate 33 cross-reactive mAbs and found that they target multiple epitopes on the E1 and E2 proteins. We identified five human bNAbs that neutralize CHIKV, MAYV, and other alphaviruses with differing potencies. Epitope binning and viral escape studies suggest that human bNAbs target related but distinct regions of the B domain of E2. Remarkably, the sequence analysis of human bNAbs showed few somatic mutations, and inferred germline variants largely retained neutralizing function. Two bNAbs demonstrated protection against both CHIKV- and MAYV-induced musculoskeletal disease in mice. Together, these studies further define heterologous humoral immunity among related alphaviruses in humans as well as the determinants of antibody-mediated cross-protection.