Our analysis found that BEN occurred in 7% of the Black South African population enrolled in HVTN 503. These participants also had lower median ANC when compared to a matched sample of Non-Hispanic Whites drawn from the general population of the US. Additionally, we were unable to demonstrate an association between BEN and HIV acquisition. However, female gender, cannabis use, and randomization to the HVTN 503 vaccine treatment arm were associated with an increased risk for HIV acquisition. Finally, no association was found between BEN and the incidence of adverse events, including infections and infestations.

BEN has been described in research conducted in populations of African, Middle Eastern and Caribbean ancestry [19,21,30]. However, few studies have been performed within the South African population. Literature has reported BEN prevalence rates of up to 50% in certain populations [14–21], but comparing results from these studies is challenging as various cut-off reference values have been used. Our study found an overall prevalence of 7% when a lower reference limit of 1.5x10⁹ cells/L was used to define neutropenia. The establishment of ANC reference ranges based on racial or ethnic differentiation has been considered by numerous authors previously [12–14]. Implementation of ethnicity-specific reference ranges for ANC might prove logistically challenging but may serve as a solution to issues encountered by the use of one reference range for various ethnicities, which may negatively affect patient management, e.g. deciding when to initiate or stop chemotherapy based on neutrophil values, or the inappropriate use of antibiotics in conditions based on a diagnosis of presumed sepsis/bacteraemia due to a benign, decreased neutrophil count [9,39,40].

Most participants with neutropenia in our analysis had ANC between 1.0–1.5x10⁹ cells/L. However, this reduction was not significantly associated with an increased incidence of infections. It is well established that leucocytes, particularly neutrophils, are vital in providing antimicrobial protection against bacteria and fungi. In individuals with acute leukaemia, the risk of severe infection was inversely proportional to the leucocyte count [41]. In addition, the risk of severe infections is greatly increased when the neutrophil count falls below 0.5 x10⁹ cells/L [42]. It has been suggested that BEN is caused by neutrophil marginalisation in the bone marrow resulting in reduced circulating neutrophils, rather than a reduction in the total number of systemic neutrophils [14,19]. The hypothesis that BEN does not result in impaired neutrophil-mediated immunity is supported by our findings, wherein we failed to find an association between reported adverse events and BEN, particularly adverse events of an infectious aetiology. A previous study that analysed gene expression levels in an African-American cohort was also unable to find a significant difference between participants with BEN and those with normal neutrophil counts, suggesting that circulating neutrophils from individuals with BEN are functionally similar to those without BEN [43]. To our knowledge, this is the first study that sought to investigate the association between BEN and the frequency or type of adverse events in a clinical trial of healthy adults. These results are reassuring that the presence of BEN is unlikely to have a significant effect on adverse event reporting in HIV vaccine clinical trials.

A large amount of variability was noted in the distribution of BEN among the various South African regions sampled. Participants from the Pretoria North region were noted to have the highest prevalence of neutropenia when compared with other regions. Interestingly, Black participants in Cape Town had a much lower prevalence of neutropenia (0.7%) when compared with Black participants from other regions. Recent literature has strongly implicated a genetic aetiology for BEN. More specifically, rs2814778, a single nucleotide polymorphism (SNP) on chromosome 1, was the strongest predictor of neutropenia in 4 recent studies that conducted genome analyses to investigate possible genetic marker associations with BEN. This SNP has also been linked with a Duffy null phenotype associated with the lack of Duffy Antigen Receptor for Chemokine (DARC) expression on erythrocyte cell membranes [16,44–46]. A visual analysis of the geographical variation of BEN in our South African cohort demonstrated a general decline in the prevalence of BEN in comparison to the northern region of South Africa, with Cape Town located in the southern region. A similar pattern has been reported in epidemiological studies that showed steep “clines” in the DARC null phenotype from the Central African belt which demonstrated prevalence rates ≥90%, to the southernmost point in Africa which had median frequencies of <10% [47,48]. Considering that this marker appears to be a strong indicator of African ancestry in the majority of regions in Sub-Saharan Africa, it is worth hypothesizing that the reduced prevalence of neutropenia in the Cape Town region may be indicative of genetic admixture. Alternatively, this observation may, at least in part, be due to a historically decreased prevalence of malaria due to Plasmodium vivax in the southern African region [16,48,49]. This is relevant as the currently accepted aetiology for BEN is that the DARC-null phenotype conferred an evolutionary advantage to infection by P. vivax, which allowed for genetic fixation in Sub-Saharan Africa [47,50,51]. Populations residing in areas with decreased P. vivax infection will, therefore, have been less likely to positively select the genetic SNP, thereby resulting in a decreased prevalence of BEN.

Research on contributory factors for BEN have previously included genetic and environmental factors, and the aetiology is likely multifactorial. The findings in this paper support this, as cannabis use and BMI were significantly associated with the prevalence of BEN. Previous murine and in-vitro data have shown that cannabinoid compounds inhibit neutrophil migration and function, which may have negative effects on the neutrophil cell count [52,53]. However, data on associations between cannabis use and BEN are scarce. This may have important consequences for patient care in therapeutic areas such as psychiatry where cannabis use is frequent and likely to become more prevalent in the current context of cannabis legalisation in South Africa [54]. Additionally, neutropenia in a psychotic patient with concomitant clozapine and cannabis use may, therefore, be incorrectly attributed to clozapine, instead of cannabis. The concomitant use of these agents may also exert additive myelosuppressive effects that further increase the patient’s risk for agranulocytosis [40,55] Further research is warranted to investigate these associations. A significant difference in BMI between participants with BEN and those with normal neutrophil counts also correlates with data that elevated neutrophil counts were associated with an increased BMI in adolescents with asthma [56]. The aetiology for this association is unclear but may either relate to an altered inflammatory state in individuals with an increased BMI, thus resulting in increased neutrophil counts, or it may be due to linkage disequilibrium between genetic polymorphisms that contribute towards an increased BMI and SNPs associated with BEN [57].

The results from our study showed that, at a lower limit of 1.5 x10⁹ cells/L, the presence of BEN did not significantly influence the participants’ susceptibility for HIV infection. Previous research is conflicting, with some studies demonstrating an increased susceptibility for HIV infection with decreased neutrophil counts [4,6], while others failed to show an association [58–60]. While the role of the adaptive immune system in the pathogenesis of HIV has been well described [61], the innate immune system’s role is less clear. Neutrophils are among the first cell lines to reach the site of infection, and several mechanisms have been proposed and described to contribute to its protective action against HIV, including phagocytosis of HIV, neutrophil extracellular trap formation, and chemokine modulation [62,63]. Various reasons may account for the contrast in results, including differences in the risk profile of the sampled population, tribe specific characteristics, or differing prevalence rates of BEN based on the reference ranges used [9]. Overall, these results provide reassurance that the inclusion of participants with BEN in HIV vaccine clinical trials is unlikely to place them at additional risk for HIV infection.

On the contrary, female individuals and those that reported cannabis use had an approximately 2-fold increase in their risk for HIV infection. While it is well described that female individuals are at a disproportionately increased risk for HIV acquisition [64], the evidence around cannabis use as a risk factor is conflicting. A study by Woody et al was unable to demonstrate an association between cannabis use and higher sexual risk [65], but a paper published in 2015 reported that marijuana use among Black men who have sex with men was associated with a significantly increased risk for HIV acquisition [66]. Similarly, another study reported that young, heterosexual adults that used cannabis had a higher risk preference, decreased intention to use HIV protective measures, and lower HIV self-efficacy—factors that increase their HIV acquisition risk [67]. It is critical that methodologies such as HIV risk reduction counselling that are performed in most HIV vaccine clinical trials should pay particular attention to the frequent use of cannabis, and explore these risks with their participants using a non-judgmental and open-minded approach.

This study had some limitations. Our data were collected from 4 of the 9 provinces in South Africa, and thus, may not be a completely representative sample of the South African population. Our analyses were limited to complete blood count data collected at enrolment, and these data, therefore, do not account for natural fluctuations in ANC. Additionally, the NHANES dataset utilized a different study design and sampling approach, which may have introduced bias, however, this was mitigated using a time- and age-matched sample from the dataset. The ethnic categorization used in the NHANES dataset also differed from the self-designated racial categories used in the HVTN 503 study, which may have influenced our results. Lastly, HVTN 503 was not powered to fully assess the effect of BEN on HIV acquisition or adverse event reporting.