High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis

Abstract

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM.IMPORTANCE Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a major target for antibodies. We report the detection of EBV gp350-specific antibodies capable of neutralizing EBV infection in vitro. The majority of gp350-directed vaccines focus on glycoproteins from lab-adapted strains, which may poorly reflect primary viral envelope diversity. We report some of the first primary gp350 sequences, noting that the gp350 host receptor binding site is remarkably stable across patients and time. However, changes in overall gene diversity were detectable during infection. Patients with higher peripheral blood viral loads in primary infection and greater changes in viral diversity generated more efficient antibodies. Our findings provide insight into the generation of functional antibodies, necessary for vaccine development.

Early Epstein-Barr Virus Genomic Diversity and Convergence toward the B95.8 Genome in Primary Infection

“…Additional studies will be helpful in discerning whether this indicates separate and discrete selection pressures operant on these genes. Certainly, envelope glycoprotein variation is subject to selection pressure by antibody activity (16), whereas latent gene variability may be driven more by CD8 T cell selective pressures.…”

“…It has been generally assumed that double-stranded DNA (dsDNA) viral genomes such as EBV are relatively stable due to the proofreading capacity of eukaryotic DNA polymerases which restrict mutation rates to approximately 10 Ϫ9 substitutions/site/year (13). However, several recent reports from our lab and others have demonstrated that even dsDNA viral genes and genomes (e.g., cytomegalovirus [CMV] and EBV) display a measurable amount of variability within an infected host at any time after infection (14)(15)(16). The origin of this variability is unknown; it could be present in the initial viral inoculum, arise early during initial rounds of infection and replication in epithelial cells or seeding of the B cell compartment, or evolve as a result of immune escape over the course of chronic infection.…”

“…The origin of this variability is unknown; it could be present in the initial viral inoculum, arise early during initial rounds of infection and replication in epithelial cells or seeding of the B cell compartment, or evolve as a result of immune escape over the course of chronic infection. In addition, different levels of variation were detected among EBV genes within similar cohorts (15,16). Sequencing full-length genomes directly from patient samples over the course of primary through persistent EBV infection is necessary to resolve these questions.…”

Vaccine Development for Epstein-Barr Virus

“…Recent sequencing of clinical isolates indicates that gp350 is less conserved than other glycoproteins important for infection (Palser et al 2015; Santpere et al 2014), likely due to pressure to evolve in response to its role as a target for cytotoxic T cells. There is little change in gp350 amino acid sequence in individuals between the time of acute infectious mononucleosis and convalescence; the few changes that do occur are located outside the CR2 binding domain (Weiss et al 2016). In general, the CR2 binding site on gp350 is highly conserved.…”

“…Antibody-dependent cellular cytotoxicity (ADCC) directed against cells expressing gp350 was not detected in sera from persons at the onset of infectious mononucleosis, but was detected in healthy EBV-seropositive persons (Xu et al 1998). Similarly, antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during the initial phase of infectious mononucleosis, but was frequently present 6 months later (Weiss et al 2016). At present it is unknown which activities mediated by antibodies are most important for protection against EBV infection or disease.…”