Differences in immune responses to SARS-CoV-2 in children and adults are linked to clinical outcomes.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is more benign in children compared with adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS coronavirus 2 (SARS-CoV-2) protects against severe disease. METHODS: Clinical outcomes, SARS-CoV-2 viral copies, and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the emergency department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse-transcription PCR. Total protein, cytokines, and anti–SARS-CoV-2 IgG and IgA were quantified in nasal fluid. RESULTS: SARS-CoV-2 copies, angiotensin-converting enzyme 2, and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with IFN signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-α2, IFN-γ, IP-10, IL-8, and IL-1β protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells, whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti–SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen, whereas 7 adults did ( P = 0.03); 4 adults died. CONCLUSION: These findings provide direct evidence of a more vigorous early mucosal immune response in children compared with adults and suggest that this contributes to favorable clinical outcomes. FUNDING: NIH grants R01 AI134367, UL1 TR002556, T32 AI007501, T32GM007288, P30 AI124414; an Albert Einstein College of Medicine Dean’s COVID-19 Pilot Research Award; and the Eric J. Heyer, MD, PhD Translational Research Pilot Project Award.

Herpes simplex virus (HSV) entry is associated with Akt translocation to the outer leaflet of the plasma membrane to promote a complex signaling cascade. We hypothesized that phospholipid scramblase-1 (PLSCR1), a calcium responsive enzyme that flips phosphatidylserines between membrane leaflets, might redistribute Akt to the outside during entry. Confocal imaging, biotinylation of membrane proteins and flow cytometric analysis demonstrated that HSV activates PLSCR1 and flips phosphatidylserines and Akt to the outside shortly following HSV-1 or HSV-2 exposure. Translocation was blocked by addition of a cell permeable calcium chelator, pharmacological scramblase antagonist, or transfection with small interfering RNA targeting PLSCR1. Co-immunoprecipitation and proximity ligation studies demonstrated that PLSCR1 associated with glycoprotein L at the outer leaflet and studies with gL deletion viruses indicate that this interaction facilitates subsequent restoration of the plasma membrane architecture. Ionomycin, a calcium ionophore, also induced PLSCR1 activation resulting in Akt externalization, suggesting a previously unrecognized biological phenomenon. Defining the mechanisms by which herpes simplex viruses (HSV) enter cells will facilitate the development of new strategies to prevent or treat infections and provide insights into cell biology. We report the novel observation that HSV activates the enzyme, scramblase, which redistributes phosphatidylserines, the major component of the inner leaflet of the plasma membrane, and the associated protein, Akt, between the inner and outer leaflet of the plasma membrane, to promote viral entry.

Herpes simplex virus type 2 (HSV-2) coinfection is associated with increased HIV-1 viral loads and expanded tissue reservoirs, but the mechanisms are not well defined. HSV-2 recurrences result in an influx of activated CD4⁺ T cells to sites of viral replication and an increase in activated CD4⁺ T cells in peripheral blood. We hypothesized that HSV-2 induces changes in these cells that facilitate HIV-1 reactivation and replication and tested this hypothesis in human CD4⁺ T cells and 2D10 cells, a model of HIV-1 latency. HSV-2 promoted latency reversal in HSV-2–infected and bystander 2D10 cells. Bulk and single-cell RNA-Seq studies of activated primary human CD4⁺ T cells identified decreased expression of HIV-1 restriction factors and increased expression of transcripts including MALAT1 that could drive HIV replication in both the HSV-2–infected and bystander cells. Transfection of 2D10 cells with VP16, an HSV-2 protein that regulates transcription, significantly upregulated MALAT1 expression, decreased trimethylation of lysine 27 on histone H3 protein, and triggered HIV latency reversal. Knockout of MALAT1 from 2D10 cells abrogated the response to VP16 and reduced the response to HSV-2 infection. These results demonstrate that HSV-2 contributes to HIV-1 reactivation through diverse mechanisms, including upregulation of MALAT1 to release epigenetic silencing.

Herpes simplex viruses (HSV) are significant global health problems associated with mucosal and neurologic disease. Prior experimental vaccines primarily elicited neutralizing antibodies targeting glycoprotein D (gD), but those that advanced to clinical efficacy trials have failed. Preclinical studies with an HSV-2 strain deleted in gD (ΔgD-2) administered subcutaneously demonstrated that it elicited a high titer, weakly neutralizing antibodies that activated Fcγ receptors to mediate antibody-dependent cellular cytotoxicity (ADCC), and completely protected mice against lethal disease and latency following vaginal or skin challenge with HSV-1 or HSV-2. Vaccine efficacy, however, may be impacted by dose and route of immunization. Thus, the current studies were designed to compare immunogenicity and efficacy following different routes of vaccination with escalating doses of ΔgD-2. We compared ΔgD-2 with two other candidates: recombinant gD protein combined with aluminum hydroxide and monophosphoryl lipid A adjuvants and a replication-defective virus deleted in two proteins involved in viral replication, dl5-29. Compared to the subcutaneous route, intramuscular and/or intradermal immunization resulted in increased total HSV antibody responses for all three vaccines and boosted the ADCC, but not the neutralizing response to ΔgD and dl5-29. The adjuvanted gD protein vaccine provided only partial protection and failed to elicit ADCC independent of route of administration. In contrast, the increased ADCC following intramuscular or intradermal administration of ΔgD-2 or dl5-29 translated into significantly increased protection. The ΔgD-2 vaccine provided 100% protection at doses as low as 5 × 10⁴ pfu when administered intramuscularly or intradermally, but not subcutaneously. However, administration of a combination of low dose subcutaneous ΔgD-2 and adjuvanted gD protein resulted in greater protection than low dose ΔgD-2 alone indicating that gD neutralizing antibodies may contribute to protection. Taken together, these results demonstrate that ADCC provides a more predictive correlate of protection against HSV challenge in mice and support intramuscular or intradermal routes of vaccination.

Herpes simplex virus (HSV) prevention is a global health priority but, despite decades of research, there is no effective vaccine. Prior efforts focused on generating glycoprotein D (gD) neutralizing antibodies, but clinical trial outcomes were disappointing. The deletion of gD yields a single-cycle candidate vaccine (∆gD-2) that elicits high titer polyantigenic non-gD antibodies that exhibit little complement-independent neutralization but mediate antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Active or passive immunization with ΔgD-2 completely protects mice from lethal disease and latency following challenge with clinical isolates of either serotype. The current studies evaluated the role of complement in vaccine-elicited protection. The immune serum from the ΔgD-2 vaccinated mice exhibited significantly greater C1q binding compared to the serum from the gD protein vaccinated mice with infected cell lysates from either serotype as capture antigens. The C1q-binding antibodies recognized glycoprotein B. This resulted in significantly greater antibody-mediated complement-dependent cytolysis and neutralization. Notably, complete protection was preserved when the ΔgD-2 immune serum was passively transferred into C1q knockout mice, suggesting that ADCC and ADCP are sufficient in mice. We speculate that the polyfunctional responses elicited by ΔgD-2 may prove more effective in preventing HSV, compared to the more restrictive responses elicited by adjuvanted gD protein vaccines.