The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is arranged as a trimer on the virus surface, composed of three S1 and three S2 subunits. Infected and vaccinated individuals generate antibodies against spike, which can neutralize the virus. Most antibodies target the receptor-binding domain (RBD) and N-terminal domain (NTD) of S1; however, antibodies against other regions of spike have also been isolated. The interhost variability in domain specificity and relative neutralization efficacy of the antibodies is still poorly characterized. To this end, we tested serum and plasma samples collected from 85 coronavirus disease 2019 (COVID-19) convalescent subjects. Samples were analyzed using seven immunoassays that employ different domains, subunits, and oligomeric forms of spike to capture the antibodies. Samples were also tested for their neutralization of pseudovirus containing SARS-CoV-2 spike and of replication-competent SARS-CoV-2. While the total amount of anti-spike antibodies produced varied among convalescent subjects, we observed an unexpectedly fixed ratio of RBD- to NTD-targeting antibodies. The relative potency of the response (defined as the measured neutralization efficacy relative to the total level of spike-targeting antibodies) also exhibited limited variation between subjects and was not associated with the overall amount of antispike antibodies produced. These studies suggest that host-to-host variation in the polyclonal response elicited against SARS-CoV-2 spike in early pandemic subjects is primarily limited to the quantity of antibodies generated rather than their domain specificity or relative neutralization potency. IMPORTANCE Infection by SARS-CoV-2 elicits antibodies against various domains of the spike protein, including the RBD and NTD of subunit S1 and against subunit S2. The antibody responses of different infected individuals exhibit different efficacies to inactivate (neutralize) the virus. Here, we show that the observed variation in the neutralizing activity of the antibody responses in COVID-19 convalescent subjects is caused by differences in the amounts of antibodies rather than their recognition properties or the potency of their antiviral activity. These findings suggest that COVID-19 vaccine strategies that focus on enhancing the overall level of the antibodies will likely elicit a more uniformly efficacious protective response.

Infection by HIV-1 elicits formation of antibodies that target the viral Env proteins and can inactivate the virus. The specific targets of these antibodies vary among infected individuals.

COVID-19 convalescent plasma (CCP) is currently administered to hospitalized COVID-19 patients under an EAU by the FDA. CCP units are selected by commercially available ELISA quantification of anti-SARS-CoV-2 (SARS2) antibodies under the rationale that higher levels of anti-SARS2 antibodies correlate with higher neutralizing capabilities and thus greater efficacy. The validity of this selection process has yet to be investigated. We analyzed serum samples from 60 convalesced donors with PCR-confirmed COVID-19. Samples were analyzed using commercial SARS2 ELISAs (DiaSorin, Euroimmun, Ortho, and Roche) and a cell-based enzyme-linked immunosorbent assay (cbELISA) that detects antibodies against the native conformation of the SARS2 spike glycoprotein (GP). Sera were also tested for neutralizing activity against wild-type SARS2 and a pseudovirus expressing the SARS2 GP. Immunoassay and neutralization values were compared to determine the precision of each immunoassay to predict differing levels of neutralizing activity, and ability of each immunoassay to identify high neutralizing activity. Results of immunoassays that apply SARS2 GP as the antigen (DiaSorin, Euroimmun, Ortho, and cbELISA) correlated well with neutralization titers measured by wild-type SARS2 or SARS2 GP-pseudotyped virus as compared with assays using other SARS2 antigens (Roche; N protein). Differences in precision of these assays were evident when samples within the top quintile or decile of immunoassay values were analyzed, with DiaSorin and Ortho assays showing superior performance in predicting neutralizing capability. We hope this approach will assist in the selection of highly neutralizing CCP units that confer greater clinical efficacy.

The error-prone replication machinery of HIV-1 continuously generates new variants of the envelope glycoproteins (Envs). Antibody selection pressures applied in the host can limit their persistence. The target specificity of antibodies elicited in different hosts varies considerably. Whether some specificities are shared and have affected the population-level evolution of Env structure is still unclear. We examined the historical changes in amino acid sequence of the gp41 fusion peptide proximal region (FPPR), which is not exposed on the Env trimer. For three FPPR positions, the residue found in the clade B ancestor was mainly replaced by alanine. However, the changes in alanine frequency at these positions between 1979 and 2016 followed different patterns; two positions maintained a historically-constant frequency whereas the third showed a gradual increase. To understand these patterns, we introduced alanine substitutions in the FPPR of primary HIV-1 strains and examined their fitness and antigenicity relative to the clade-ancestral form. The evolutionary patterns could not be explained by effects on Env fitness. Instead, the FPPR variants with a historically-constant alanine frequency exhibited a unique open-at-the-base conformation of the trimer that exposes partially-cryptic epitopes. These Envs were modestly but significantly more sensitive to poorly-neutralizing sera from HIV-infected individuals than the clade-ancestral form. Our findings suggest that weakly-neutralizing antibodies targeting the base of the trimer are commonly elicited. Such low-level antibody pressures do not exert catastrophic effects on the emerging variants but rather determine their set-point frequency in the population and historical patterns of change. IMPORTANCE HIV-1 infection elicits antibodies that target the Env proteins of the virus. The specific targets of these antibodies vary between infected individuals. It is unclear whether some target specificities are shared between the antibody responses of different individuals. Our data suggest that antibodies against the base of the Env protein are commonly elicited during infection and are contained in sera with low neutralization efficacy. Such antibody pressures are weak. As a result, they do not completely eliminate the sensitive Env forms from the population, but rather maintain their frequency at a low level that has not increased during the past 40 years.

Mesenchymal stromal cells (MSCs) are administered locally to treat sites of inflammation. Local delivery is known to cause MSCs to aggregate into “spheroids,” which alters gene expression and phenotype. While adherent MSCs are highly efficient in their inhibition of T cells, whether or not this property is altered upon MSC aggregation has not been thoroughly determined. In this study, we discovered that aggregation of MSCs into spheroids causes them to lose their T cell-suppressive abilities. Interestingly, adding budesonide, a topical glucocorticoid steroid, alongside spheroids partially restored MSC suppression of T cell proliferation. Through a series of inhibition and add-back studies, we determined budesonide acts synergistically with spheroid MSC-produced PGE2 to suppress T cell proliferation through the PGE2 receptors EP2 and EP4. These findings highlight critical differences between adherent and spheroid MSC interactions with human immune cells that have significant translational consequences. In addition, we uncovered a mechanism through which spheroid MSC suppression of T cells can be partly restored. By understanding the phenotypic changes that occur upon MSC aggregation and the impact of MSC drug interactions, improved immunosuppressive MSC therapies for localized delivery can be designed.

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon γ (IFNγ) exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role that the dose of IFNγ, the duration of pre-licensing and the donor of origin play in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of IFNγ and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Human mesenchymal stromal cells (MSCs) are a leading cell therapy candidate for the treatment of immune and inflammatory diseases due to their potent regulation of immune cells. MSC expression of indoleamine-2,3-dioxygenase (IDO) upon interferon gamma exposure has been proposed as both a sentinel marker and key mediator of MSC immunomodulatory potency. Rather than wait for in vivo exposure to cytokines, MSCs can be pre-licensed during manufacturing to enhance IDO expression. In this study, we systematically examine the relative role the dose of interferon gamma, the duration of pre-licensing, and the donor of origin plays in dictating MSC production of functional IDO. We find that across three human MSC donors, MSCs increase their expression of IDO in response to both increased dose of interferon gamma and duration of pre-licensing. However, with extended pre-licensing, the expression of IDO no longer predicts MSCs ability to suppress activated peripheral blood mononuclear cells. In addition, pre-licensing dose and duration are revealed to be minor modifiers of MSCs inherent potency, and thus cannot be manipulated to boost poor donors to the levels of high-performing donors. Thus, the dose and duration of pre-licensing should be tailored to optimize performance of specific donors and an emphasis on donor selection is needed to realize significant benefits of pre-licensing.

The use of mesenchymal stromal cell (MSC) therapy for the treatment of type 2 diabetes (T2D) and T2D complications is promising; however, the investigation of MSC function in the setting of T2D has not been thoroughly explored. In our current study, we investigated the phenotype and function of MSCs in a simulated in vitro T2D environment. We show that palmitate, but not glucose, exposure impairs MSC metabolic activity with moderate increases in apoptosis, while drastically affecting proliferation and morphology. In co-culture with peripheral blood mononuclear cells (PBMCs), we found that MSCs not only lose their normal suppressive ability in high levels of palmitate, but actively support and enhance inflammation, resulting in elevated PBMC proliferation and pro-inflammatory cytokine release. The pro-inflammatory effect of MSCs in palmitate was partially reversed via palmitate removal and fully reversed through pre-licensing MSCs with interferon-gamma and tumor necrosis factor alpha. Thus, palmitate, a specific metabolic factor enriched within the T2D environment, is a potent modulator of MSC immunosuppressive function, which may in part explain the depressed potency observed in MSCs isolated from T2D patients. Importantly, we have also identified a robust and durable pre-licensing regimen that protects MSC immunosuppressive function in the setting of T2D.