Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%–30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2–3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.

Sustained phosphotinositide3-kinase (PI3K) signaling is critical to the maintenance of alpha and beta herpesvirus latency. We have previously shown that the beta-herpesvirus, human cytomegalovirus (CMV), regulates epidermal growth factor receptor (EGFR), upstream of PI3K, to control states of latency and reactivation. How signaling downstream of EGFR is regulated and how this impacts CMV infection and latency is not fully understood. We demonstrate that CMV downregulates EGFR early in the productive infection, which blunts the activation of EGFR and its downstream pathways in response to stimuli. However, CMV infection sustains basal levels of EGFR and downstream pathway activity in the context of latency in CD34+ hematopoietic progenitor cells (HPCs). Inhibition of MEK/ERK, STAT or PI3K/AKT pathways downstream of EGFR increases viral reactivation from latently infected CD34+ HPCs, defining a role for these pathways in latency. We hypothesized that CMV modulation of EGFR signaling might impact viral transcription important to latency. Indeed, EGF-stimulation increased expression of the UL138 latency gene, but not immediate early or early viral genes, suggesting that EGFR signaling promotes latent gene expression. The early growth response-1 (EGR1) transcription factor is induced downstream of EGFR signaling through the MEK/ERK pathway and is important for the maintenance of hematopoietic stemness. We demonstrate that EGR1 binds the viral genome upstream of UL138 and is sufficient to promote UL138 expression. Further, disruption of EGR1 binding upstream of UL138 prevents the establishment of latency in CD34+ HPCs. Our results indicate a model whereby UL138 modulation of EGFR signaling feeds back to promote UL138 gene expression and suppression of replication for latency. By this mechanism, the virus has hardwired itself into host cell biology to sense and respond to changes in homeostatic host cell signaling.

Delineating the key early events that lead to the development of broadly neutralizing anti-HIV-1 antibodies during natural infection may help guide the development of immunogens and vaccine regimens to prevent HIV-1 infection. In this study, we monitored two HIV-1-positive subjects, VC20013 and VC10014, over the course of infection from before they developed broadly neutralizing antibody (bNAb) activity until several years after neutralizing breadth was detected in plasma. Both subjects developed bNAb activity after approximately 1 year postinfection, which ultimately mapped to the membrane-proximal external region (MPER) in VC20013 and an epitope that overlaps the CD4 receptor binding site in VC10014. In subject VC20013, we were able to identify anti-MPER activity in the earliest plasma sample that exhibited no bNAb activity, indicating that this epitope specificity was acquired very early on, but that it was initially not able to mediate neutralization. Escape mutations within the bNAb epitopes did not arise in the circulating envelopes until bNAb activity was detectable in plasma, indicating that this early response was not sufficient to drive viral escape. As bNAb activity began to emerge in both subjects, we observed a simultaneous increase in autologous antienvelope antibody binding affinity, indicating that antibody maturation was occurring as breadth was developing. Our findings illustrate one potential mechanism by which bNAbs develop during natural infection in which an epitope target is acquired very early on during the course of infection but require time and maturation to develop into broadly neutralizing activity. IMPORTANCEOne major goal of HIV-1 vaccine research is the development of a vaccine that can elicit broadly neutralizing antibodies (bNAbs). Although no such vaccine exists, bNAbs develop in approximately 20% of HIV-1-infected subjects, providing a prototype of the bNAbs that must be reelicited by vaccine. Thus, there is significant interest in understanding the mechanisms by which bNAbs develop during the course of infection. We studied the timing, epitope specificity, and evolution of the bNAb responses in two HIV-1-positive patients who developed bNAb activity within the first several years after infection. In one subject, antibodies to a broadly neutralizing epitope developed very early but were nonneutralizing. After several months, neutralizing activity developed, and the virus mutated to escape their activity. Our study highlights one mechanism for the development of bNAbs where early epitope acquisition followed by sufficient time for antibody maturation drives the epitope-specific antibody response toward broadly neutralizing activity.

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.