The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained genetically stable during the first 3 months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early-occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. IMPORTANCE The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions. The Alpha and Delta variants, which spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic compared to the original strain. Interestingly, Alpha and Delta both carried the mutations P681H/R in a cleavage site that made the spike more cleaved and more efficient at mediating viral fusion. We show here that variants with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of the D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite for the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

Targeting human langerin promotes HIV-1 specific humoral immune responses.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

Role of CD4+ T Cells in the Control of Viral Infections: Recent Advances and Open Questions.

CD4+ T cells orchestrate adaptive immune responses through their capacity to recruit and provide help to multiple immune effectors, in addition to exerting direct effector functions. CD4+ T cells are increasingly recognized as playing an essential role in the control of chronic viral infections. In this review, we present recent advances in understanding the nature of CD4+ T cell help provided to antiviral effectors. Drawing from our studies of natural human immunodeficiency virus (HIV) control, we then focus on the role of high-affinity T cell receptor (TCR) clonotypes in mediating antiviral CD4+ T cell responses. Last, we discuss the role of TCR affinity in determining CD4+ T cell differentiation, reviewing the at times divergent studies associating TCR signal strength to the choice of a T helper 1 (Th1) or a T follicular helper (Tfh) cell fate.

Triggering of TLR-3, -4, NOD2, and DC-SIGN reduces viral replication and increases T-cell activation capacity of HIV-infected human dendritic cells.

A variety of signals influence the capacity of dendritic cells (DCs) to mount potent antiviral cytotoxic T-cell (CTL) responses. In particular, innate immune sensing by pathogen recognition receptors, such as TLR and C-type lectines, influences DC biology and affects their susceptibility to HIV infection. Yet, whether the combined effects of PPRs triggering and HIV infection influence HIV-specific (HS) CTL responses remain enigmatic. Here, we dissect the impact of innate immune sensing by pathogen recognition receptors on DC maturation, HIV infection, and on the quality of HS CTL activation. Remarkably, ligand-driven triggering of TLR-3, -4, NOD2, and DC-SIGN, despite reducing viral replication, markedly increased the capacity of infected DCs to stimulate HS CTLs. This was exemplified by the diversity and the quantity of cytokines produced by HS CTLs primed by these DCs. Infecting DCs with viruses harboring members of the APOBEC family of antiviral factors enhanced the antigen-presenting skills of infected DCs. Our results highlight the tight interplay between innate and adaptive immunity and may help develop innovative immunotherapies against viral infections.

Divergent adaptive immune responses define two types of long COVID.

Background: The role of adaptive immune responses in long COVID remains poorly understood, with contrasting hypotheses suggesting either an insufficient antiviral response or an excessive immune response associated with inflammatory damage. To address this issue, we set to characterize humoral and CD4+ T cell responses in long COVID patients prior to SARS-CoV-2 vaccination. Methods: Long COVID patients who were seropositive (LC+, n=28) or seronegative (LC-, n=23) by spike ELISA assay were recruited based on (i) an initial SARS-CoV-2 infection documented by PCR or the conjunction of three major signs of COVID-19 and (ii) the persistence or resurgence of at least 3 symptoms for over 3 months. They were compared to COVID patients with resolved symptoms (RE, n=29) and uninfected control individuals (HD, n=29). Results: The spectrum of persistent symptoms proved similar in both long COVID groups, with a trend for a higher number of symptoms in the seronegative group (median=6 vs 4.5; P=0.01). The use a highly sensitive S-flow assay enabled the detection of low levels of SARS-CoV-2 spike-specific IgG in 22.7% of ELISA-seronegative long COVID (LC-) patients. In contrast, spike-specific IgG levels were uniformly high in the LC+ and RE groups. Multiplexed antibody analyses to 30 different viral antigens showed that LC- patients had defective antibody responses to all SARS-CoV-2 proteins tested but had in most cases preserved responses to other viruses. A sensitive primary T cell line assay revealed low but detectable SARS-CoV-2-specific CD4 responses in 39.1% of LC- patients, while response frequencies were high in the LC+ and RE groups. Correlation analyses showed overall strong associations between humoral and cellular responses, with exceptions in the LC- group. Conclusions: These findings provide evidence for two major types of antiviral immune responses in long COVID. Seropositive patients showed coordinated cellular and humoral responses at least as high as those of recovered patients. In contrast, ELISA-seronegative long COVID patients showed overall low antiviral responses, with detectable specific CD4+ T cells and/or antibodies in close to half of patients (52.2%). These divergent findings in patients sharing a comparable spectrum of persistent symptoms raise the possibility of multiple etiologies in long COVID.

Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry.

HIV elite controllers maintain a population of CD4 + T cells endowed with high avidity for Gag antigens and potent effector functions. How these HIV-specific cells avoid infection and depletion upon encounter with the virus remains incompletely understood. Ex vivo characterization of single Gag-specific CD4 + T cells reveals an advanced Th1 differentiation pattern in controllers, except for the CCR5 marker, which is downregulated compared to specific cells of treated patients. Accordingly, controller specific CD4 + T cells show decreased susceptibility to CCR5-dependent HIV entry. Two controllers carried biallelic mutations impairing CCR5 surface expression, indicating that in rare cases CCR5 downregulation can have a direct genetic cause. Increased expression of ß-chemokine ligands upon high-avidity antigen/TCR interactions contributes to autocrine CCR5 downregulation in controllers without CCR5 mutations. These findings suggest that genetic and functional regulation of the primary HIV coreceptor CCR5 play a key role in promoting natural HIV control.

DC Subsets Regulate Humoral Immune Responses by Supporting the Differentiation of Distinct Tfh Cells.

To determine the contribution of skin DC subsets in the regulation of humoral immunity, we used a well-characterized antigen targeting system to limit antigen availability and presentation to certain skin-derived DC subsets. Here we show that delivery of foreign antigen to steady state Langerhans cells (LCs) and cDC1s through the same receptor (Langerin) led to, respectively, robust vs. minimal-to-null humoral immune response. LCs, unlike cDC1s, supported the formation of germinal center T follicular helper cells (GC-Tfh) antigen dose-dependently and then, likely licensed by these T cells, some of the LCs migrated to the B cell area to initiate B cell responses. Furthermore, we found that the cDC1s, probably through their superior T cell activation capacity, prevented the LCs from inducing GC-Tfh cells and humoral immune responses. We further show that targeted delivery of cytokines to DCs can be used to modulate DC-induced humoral immune responses, which has important therapeutic potential. Finally, we show that human LCs, unlike monocyte-derived DCs, can support GC Tfh generation in an in vitro autologous system; and in agreement with mouse data, we provide evidence in NHP studies that targeting LCs without adjuvants is an effective way to induce antibody responses, but does not trigger CD8+ T cell responses. Our findings suggest that the major limitations of some relatively ineffective vaccines currently in use or in development might be that (1) they are not formulated to specifically target a certain subset of DCs and/or (2) the antigen dose is not tailored to maximize the intrinsic/pre-programmed capabilities of the specific DC subset. This new and substantial departure from the status quo is expected to overcome problems that have hindered our ability to generate effective vaccines against some key pathogens.