The Intact Proviral DNA Assay (IPDA) was developed to address the critical need for a scalable method for intact HIV-1 reservoir quantification. This droplet digital PCR-based assay simultaneously targets two HIV-1 regions to distinguish genomically intact proviruses against a large background of defective ones, and its application has yielded insights into HIV-1 persistence. Reports of assay failures however, attributed to HIV-1 polymorphism, have recently emerged. Here, we describe a diverse North American cohort of people with HIV-1 subtype B, where the IPDA yielded a failure rate of 28% due to viral polymorphism. We further demonstrate that within-host HIV-1 diversity can lead the IPDA to underestimate intact reservoir size, and provide examples of how this phenomenon could lead to erroneous interpretation of clinical trial data. While the IPDA represents a major methodological advance, HIV-1 diversity should be addressed before its widespread adoption as a principal readout in HIV-1 remission trials.

were used immediately if possible or cryopreserved in liquid nitrogen; cells were not left in culture before the initiation of experiments.

Antiretroviral therapies (ARTs) abrogate HIV replication; however, infection persists as long-lived reservoirs of infected cells with integrated proviruses, which reseed replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in reducing viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle: by querying the dynamics of HIV-specific T cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. In longitudinal assessments, we show that the rates of change in persisting HIV Nef-specific responses, but not responses to other HIV gene products, were associated with residual frequencies of infected cells. These Nef-specific responses were highly stable over time and disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV antigens. These results indicate substantial visibility of the HIV-infected cells to T cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing infection.

Lost to follow-up (n = 0) Lost to follow-up (n = 1) Lost to follow-up (n = 2) Moved out of the study area (n = 1) Due to a SAE (n = 1) Lost to follow-up (n = 0) Analyses were performed as indicated above and as detailed in the comprehensive CONSORT flow diagram presented in Extended Data Fig. 1. Enrollment Allocation Follow-up Analysis HIV-1 mRNA + and/or p24 + cells (FISH-flow)HIV-1-specific immunity (AIM)

Resistance is a key determinant in interactions between hosts and their parasites. Understanding the amount and distribution of variation in this trait between strains can provide insights into (co)evolutionary processes and their potential to shape patterns of diversity in natural populations. Using controlled inoculation in experimental mass cultures, we investigated the quantitative variation in resistance to the bacterial parasite Holospora undulata across a worldwide collection of strains of its ciliate host Paramecium caudatum. We combined the observed variation with available information on the phylogeny and biogeography of the strains. We found substantial variation in resistance among strains, with upper-bound values of broad-sense heritability >0.5 (intraclass correlation coefficients). Strain estimates of resistance were repeatable between laboratories and ranged from total resistance to near-complete susceptibility. Early (1 week post inoculation) measurements provided higher estimates of resistance heritability than did later measurements (2–3 weeks), possibly due to diverging epidemiological dynamics in replicate cultures of the same strains. Genetic distance (based on a neutral marker) was positively correlated with the difference in resistance phenotype between strains (r = 0.45), essentially reflecting differences between highly divergent clades (haplogroups) within the host species. Haplogroup A strains, mostly European, were less resistant to the parasite (49% infection prevalence) than non-European haplogroup B strains (28%). At a smaller geographical scale (within Europe), strains that are geographically closer to the parasite origin (Southern Germany) were more susceptible to infection than those from further away. These patterns are consistent with a picture of local parasite adaptation. Our study demonstrates ample natural variation in resistance on which selection can act and hints at symbiont adaptation producing signatures in geographic and lineage-specific patterns of resistance in this model system.