Too risky to use, or too risky not to? Lessons learned from over 30 years of research on forensic risk assessment with Indigenous persons.

Indigenous peoples are overrepresented in correctional systems internationally, reflecting a history of systemic racism and colonial oppression, and the practice of risk assessment with this population has been a focus of legal and sociopolitical controversy. We conducted a systematic review and meta-analysis of the risk assessment literature comparing Indigenous and non-Indigenous (White majority) groups. We retrieved 91 studies featuring 22 risk tools and 15 risk/need/cultural domains (N = 59,693, Indigenous; N = 237,729, non-Indigenous/White) and four documents identifying culturally relevant factors. Most measures demonstrated moderate predictive validity but often had significant ethnoracial differences, particularly for static measures. The Service Planning Instrument/Youth Assessment Screening Inventory, Level of Service Inventory youth variants, Psychopathy Checklist-Revised and Youth Version, and the Violence Risk Scale and its Sexual Offense version had the strongest predictive validity and least ethnoracial discrepancy. The Static Factors Assessment and Dynamic Factors Identification and Analysis-Revised had the weakest predictive validity. For Indigenous persons, the strongest individual predictors of recidivism were low education/employment, substance abuse, antisocial pattern, and poor community functioning, while mitigating factors that predicted decreased recidivism were measures of risk change (i.e., from culturally integrated programs combining mainstream and traditional healing approaches), cultural engagement/connectedness, and protective factors. In practice, static measures need to be supplemented with dynamic ones, and assessors should select measures with at least moderate predictive validity and ideally the least ethnoracial bias. These conclusions are tempered by the quantity and quality of the literature coupled with the circumstance that some study authors have coauthored tools in this review. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Mixed method examination of alcohol and suicidality among actively suicidal adults who engage in heavy episodic drinking.

Suicide is a serious public health problem in the United States. Alcohol use has been substantially documented as a risk factors for suicide, yet it is unclear how alcohol is associated with suicidal ideation (SI) and behavior (SIB) at the event level. We examined the association between alcohol use and SI using a mixed methods approach that included daily assessments from 13 adults who engage in heavy episodic drinking with current SI and qualitative interviews among 12 of those adults. Participants were recruited on social media. Separate mixed effects logistic regression models indicated that individuals' alcohol use on a given day was associated with SI (OR = 1.37), and suicidal urges (OR = 1.41). Adjusting for repeated measures, the expected marginal mean for intensity of SI (EMM = 3.33) and urges (EMM = 2.94) were higher on days with reported drinking behavior than days without reported drinking (EMM = 2.68 and EMM = 2.62 respectively). Qualitative data indicated that the association between alcohol use and SIB is more complex than a single directionality. Instead, the association can be unidirectional, bidirectional, and/or dependent on factors including mental health and amount of alcohol consumed. Overall, these findings emphasize a need for integrated alcohol and SIB interventions while providing insight on possible daily, just-in-time adaptations.

Racial Disparities in Sexual Assault Characteristics and Mental Health Care After Sexual Assault Medical Forensic Exams.

Background: Sexual assault (SA) is common, but Black individuals might be at higher risk of SA and negative health sequalae. Racial differences in SA characteristics and health care utilization after SA are largely unknown. Materials and Methods: We reviewed medical records of 690 individuals (23.9% Black; 93.6% women) who received a SA medical forensic exam (SAMFE) at a southeastern U.S. hospital. We examined bivariate racial differences in SA characteristics and used zero-inflated Poisson regressions to estimate racial differences in mental health outpatient visits at the SAMFE hospital. Results: Among survivors of SA, Black survivors were more likely than White survivors to have been victimized by an intimate partner (odds ratio [OR] = 1.77, confidence interval [95% CI] = 1.02-3.07) and they had more post-SA outpatient mental health visits at the SAMFE hospital (incidence rate ratio [IRR] = 2.05, 95% CI = 1.70-2.47). Black survivors were less likely to report alcohol or drug use before the SA (OR = 0.42, 95% CI = 0.28-0.62). In multivariable models, Black survivors trended toward more mental health visits than White survivors (IRR = 1.63, 95% CI = 0.82-2.44), but intimate partner violence (IPV) significantly moderated that association (IRR = 0.01, 95%CI = ≤0.001-0.03). Black survivors assaulted by an intimate partner were less likely to access mental health care than White IPV survivors. Conclusions: The hospital setting of a SAMFE could be a unique opportunity to serve Black survivors and reduce racial disparities in mental health sequelae, but additional support will be needed for Black survivors experiencing IPV. An intersectional, reproductive justice framework has the potential to address these challenges.

Radiosensitisation of Hepatocellular Carcinoma Cells by Vandetanib.

Hepatocellular Carcinoma (HCC) is increasing in incidence worldwide and requires new approaches to therapy. The combination of anti-angiogenic drug therapy and radiotherapy is one promising new approach. The anti-angiogenic drug vandetanib is a tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and RET proto-oncogene with radio-enhancement potential. To explore the benefit of combined vandetanib and radiotherapy treatment for HCC, we studied outcomes following combined treatment in pre-clinical models. METHODS: Vandetanib and radiation treatment were combined in HCC cell lines grown in vitro and in vivo. In addition to 2D migration and clonogenic assays, the combination was studied in 3D spheroids and a syngeneic mouse model of HCC. RESULTS: Vandetanib IC 50 s were measured in 20 cell lines and the drug was found to significantly enhance radiation cell kill and to inhibit both cell migration and invasion in vitro. In vivo, combination therapy significantly reduced cancer growth and improved overall survival, an effect that persisted for the duration of vandetanib treatment. CONCLUSION: In 2D and 3D studies in vitro and in a syngeneic model in vivo, the combination of vandetanib plus radiotherapy was more efficacious than either treatment alone. This new combination therapy for HCC merits evaluation in clinical trials.

Protective HLA alleles are associated with reduced LPS levels in acute HIV infection with implications for immune activation and pathogenesis.

Despite extensive research on the mechanisms of HLA-mediated immune control of HIV-1 pathogenesis, it is clear that much remains to be discovered, as exemplified by protective HLA alleles like HLA-B*81 which are associated with profound protection from CD4+ T cell decline without robust control of early plasma viremia. Here, we report on additional HLA class I (B*1401, B*57, B*5801, as well as B*81), and HLA class II (DQB1*02 and DRB1*15) alleles that display discordant virological and immunological phenotypes in a Zambian early infection cohort. HLA class I alleles of this nature were also associated with enhanced immune responses to conserved epitopes in Gag. Furthermore, these HLA class I alleles were associated with reduced levels of lipopolysaccharide (LPS) in the plasma during acute infection. Elevated LPS levels measured early in infection predicted accelerated CD4+ T cell decline, as well as immune activation and exhaustion. Taken together, these data suggest novel mechanisms for HLA-mediated immune control of HIV-1 pathogenesis that do not necessarily involve significant control of early viremia and point to microbial translocation as a direct driver of HIV-1 pathogenesis rather than simply a consequence.

Frontline Science: Late CD27 stimulation promotes IL-7Rα transcriptional re-expression and memory T cell qualities in effector CD8+ T cells.

We previously demonstrated that CD27 co-stimulation during a primary CD8+ T-cell response was critical for the expression of IL-7Rα on acute effector CD8+ T cells, providing an essential element in the generation of CD8+ T-cell memory to infectious pathogens. IL-7 plays a critical role in the generation and maintenance of memory CD8+ T cells, and IL-7Rα has been regarded as a functional marker of long-lived memory precursor effector cells. While IL-7Rα is downregulated acutely upon TCR stimulation, the regulation of the emergence of IL-7Rα expressing cells around the peak of primary CD8+ responses is less clear. Re-expression could be a default outcome after withdrawal of TCR stimulation. Alternatively, specific stimuli could actively antagonize the downregulation or promote the recovery of IL-7Rα in Ag-activated CD8+ T cells. By utilizing agonistic mAb and transgenic models, here we show: (1) CD27 stimulation acts directly on CD8+ T cells to enhance IL-7Rα-expressing effectors; (2) CD27 stimulation neither alleviates the downregulation of IL-7Rα upon TCR signaling nor promotes the expansion/survival of IL-7Rα-expressing effectors, but facilitates IL-7Rα re-expression; (3) CD27 stimulation regulates Il7ra mRNA abundance but not protein distribution. Importantly, CD27 stimulation promotes not only IL-7Rα, but also the common γ chain of the receptor and the downstream signaling mediated by pSTAT5. Our results demonstrate a previously unappreciated role of CD27 stimulation as a positive regulator of IL-7Rα during CD8 T-cell responses, provide insights into the mechanistic basis by which CD27 stimulation influences CD8+ T-cell memory differentiation, and highlight the potential of targeting CD27-CD70 axis to enhance IL-7 signaling for antiviral/antitumor immunotherapy.

Diacylglycerol Lipase-ß Is Required for TNF-α Response but Not CD8+ T Cell Priming Capacity of Dendritic Cells.

Diacylglycerol lipase-ß (DAGLß) hydrolyzes arachidonic acid (AA)-esterified diacylglycerols to produce 2-arachidonoylglycerol (2-AG) and downstream prostanoids that mediate inflammatory responses of macrophages. Here, we utilized DAGL-tailored activity-based protein profiling and genetic disruption models to discover that DAGLß regulates inflammatory lipid and protein signaling pathways in primary dendritic cells (DCs). DCs serve as an important link between innate and adaptive immune pathways by relaying innate signals and antigen to drive T cell clonal expansion and prime antigen-specific immunity. We discovered that disruption of DAGLß in DCs lowers cellular 2-AG and AA that is accompanied by reductions in lipopolysaccharide (LPS) stimulated tumor necrosis factor α secretion. Cell-based vaccination studies revealed that DC maturation ex vivo and immunogenicity in vivo was surprisingly unaffected by DAGLß inactivation. Collectively, we identify DAGLß pathways as a means for attenuating DC inflammatory signaling while sparing critical adaptive immune functions and further expand the utility of targeting lipid pathways for immunomodulation.

Small bowel adenocarcinoma in a patient with Lynch syndrome.

A 49-year-old male patient, morbidly obese, with a background of Lynch syndrome and subtotal colectomy for colon cancer in 2007, presented with severe abdominal pain in December 2015. Since then, the patient presented multiple times to the emergency department with severe diffuse abdominal pain. After extensive examination, no clear cause for the pain was identified and it was thought to be secondary to adhesions, incisional hernias and psychological. Examinations via radiological imaging were challenging due to body habitus and claustrophobia. In September 2017, the patient was admitted from outpatient clinic with severe abdominal pain, weight loss and anaemia. A CT scan of abdomen and pelvis demonstrated a dilated jejunal loop with a possible tumour. Surgery confirmed a small bowel tumour and, nearly 2 years after the initial presentation, the patient was diagnosed with adenocarcinoma of the jejenum. The patient underwent surgical excision and his symptoms subsided.

HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs.

HIV-1 downregulates human leukocyte antigen A (HLA-A) and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing natural killer (NK) cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B, and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C, and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3- to 6.1-fold) which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to in vitro NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B, and HLA-C exist within and between individuals that can have functional consequences on immune recognition.IMPORTANCE Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome often are not used in experimental systems due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of HLA class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that the HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.

Wide variation in susceptibility of transmitted/founder HIV-1 subtype C Isolates to protease inhibitors and association with in vitro replication efficiency.

The gag gene is highly polymorphic across HIV-1 subtypes and contributes to susceptibility to protease inhibitors (PI), a critical class of antiretrovirals that will be used in up to 2 million individuals as second-line therapy in sub Saharan Africa by 2020. Given subtype C represents around half of all HIV-1 infections globally, we examined PI susceptibility in subtype C viruses from treatment-naïve individuals. PI susceptibility was measured in a single round infection assay of full-length, replication competent MJ4/gag chimeric viruses, encoding the gag gene and 142 nucleotides of pro derived from viruses in 20 patients in the Zambia-Emory HIV Research Project acute infection cohort. Ten-fold variation in susceptibility to PIs atazanavir and lopinavir was observed across 20 viruses, with EC50s ranging 0.71-6.95 nM for atazanvir and 0.64-8.54 nM for lopinavir. Ten amino acid residues in Gag correlated with lopinavir EC50 (p < 0.01), of which 380 K and 389I showed modest impacts on in vitro drug susceptibility. Finally a significant relationship between drug susceptibility and replication capacity was observed for atazanavir and lopinavir but not darunavir. Our findings demonstrate large variation in susceptibility of PI-naïve subtype C viruses that appears to correlate with replication efficiency and could impact clinical outcomes.

Balance between transmitted HLA preadapted and nonassociated polymorphisms is a major determinant of HIV-1 disease progression.

HIV-1 adapts to a new host through mutations that facilitate immune escape. Here, we evaluate the impact on viral control and disease progression of transmitted polymorphisms that were either preadapted to or nonassociated with the new host's HLA. In a cohort of 169 Zambian heterosexual transmission pairs, we found that almost one-third of possible HLA-linked target sites in the transmitted virus Gag protein are already adapted, and that this transmitted preadaptation significantly reduced early immune recognition of epitopes. Transmitted preadapted and nonassociated polymorphisms showed opposing effects on set-point VL and the balance between the two was significantly associated with higher set-point VLs in a multivariable model including other risk factors. Transmitted preadaptation was also significantly associated with faster CD4 decline (<350 cells/µl) and this association was stronger after accounting for nonassociated polymorphisms, which were linked with slower CD4 decline. Overall, the relative ratio of the two classes of polymorphisms was found to be the major determinant of CD4 decline in a multivariable model including other risk factors. This study reveals that, even before an immune response is mounted in the new host, the balance of these opposing factors can significantly influence the outcome of HIV-1 infection.

Genomic Modifiers of Natural Killer Cells, Immune Responsiveness and Lymphoid Tissue Remodeling Together Increase Host Resistance to Viral Infection.

The MHC class I D(k) molecule supplies vital host resistance during murine cytomegalovirus (MCMV) infection. Natural killer (NK) cells expressing the Ly49G2 inhibitory receptor, which specifically binds D(k), are required to control viral spread. The extent of D(k)-dependent host resistance, however, differs significantly amongst related strains of mice, C57L and MA/My. As a result, we predicted that relatively small-effect modifier genetic loci might together shape immune cell features, NK cell reactivity, and the host immune response to MCMV. A robust D(k)-dependent genetic effect, however, has so far hindered attempts to identify additional host resistance factors. Thus, we applied genomic mapping strategies and multicolor flow cytometric analysis of immune cells in naive and virus-infected hosts to identify genetic modifiers of the host immune response to MCMV. We discovered and validated many quantitative trait loci (QTL); these were mapped to at least 19 positions on 16 chromosomes. Intriguingly, one newly discovered non-MHC locus (Cmv5) controlled splenic NK cell accrual, secondary lymphoid organ structure, and lymphoid follicle development during MCMV infection. We infer that Cmv5 aids host resistance to MCMV infection by expanding NK cells needed to preserve and protect essential tissue structural elements, to enhance lymphoid remodeling and to increase viral clearance in spleen.

Replicative fitness of transmitted HIV-1 drives acute immune activation, proviral load in memory CD4+ T cells, and disease progression.

HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the factors that influence HIV-1-induced immunopathology and subsequent CD4(+) T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8(+) T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4(+) T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1-related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.

Transmitted virus fitness and host T cell responses collectively define divergent infection outcomes in two HIV-1 recipients.

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.

A restriction enzyme based cloning method to assess the in vitro replication capacity of HIV-1 subtype C Gag-MJ4 chimeric viruses.

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

HIV transmission. Selection bias at the heterosexual HIV-1 transmission bottleneck.

Heterosexual transmission of HIV-1 typically results in one genetic variant establishing systemic infection. We compared, for 137 linked transmission pairs, the amino acid sequences encoded by non-envelope genes of viruses in both partners and demonstrate a selection bias for transmission of residues that are predicted to confer increased in vivo fitness on viruses in the newly infected, immunologically naïve recipient. Although tempered by transmission risk factors, such as donor viral load, genital inflammation, and recipient gender, this selection bias provides an overall transmission advantage for viral quasispecies that are dominated by viruses with high in vivo fitness. Thus, preventative or therapeutic approaches that even marginally reduce viral fitness may lower the overall transmission rates and offer long-term benefits even upon successful transmission.

Multiparametric analysis of host response to murine cytomegalovirus in MHC class I-disparate mice reveals primacy of Dk-licensed Ly49G2+ NK cells in viral control.

MHC class I D(k) and Ly49G2 (G2) inhibitory receptor-expressing NK cells are essential to murine CMV (MCMV) resistance in MA/My mice. Without D(k), G2(+) NK cells in C57L mice fail to protect against MCMV infection. As a cognate ligand of G2, D(k) licenses G2(+) NK cells for effector activity. These data suggested that D(k)-licensed G2(+) NK cells might recognize and control MCMV infection. However, a role for licensed NK cells in viral immunity is uncertain. We combined classical genetics with flow cytometry to visualize the host response to MCMV. Immune cells collected from individuals of a diverse cohort of MA/My × C57L offspring segregating D(k) were examined before infection and postinfection, including Ly49(+) NK subsets, receptor expression features, and other phenotypic traits. To identify critical NK cell features, automated analysis of 110 traits was performed in R using the Pearson correlation, followed with a Bonferroni correction for multiple tests. Hierarchical clustering of trait associations and principal component analyses were used to discern shared immune response and genetic relationships. The results demonstrate that G2 expression on naive blood NK cells was predictive of MCMV resistance. However, rapid G2(+) NK cell expansion following viral exposure occurred selectively in D(k) offspring; this response was more highly correlated with MCMV control than all other immune cell features. We infer that D(k)-licensed G2(+) NK cells efficiently detected missing-self MHC cues on viral targets, which elicited cellular expansion and target cell killing. Therefore, MHC polymorphism regulates licensing and detection of viral targets by distinct subsets of NK cells required in innate viral control.

Role of transmitted Gag CTL polymorphisms in defining replicative capacity and early HIV-1 pathogenesis.

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone.