Antigen-presenting cells (APCs) of the myeloid lineage trigger innate and adaptive immune responses against invading viruses, thus modulating the outcome, progression, and clearance of infections (1, 2). Yet, chronic viral infections counteract several defenses orchestrated by APCs and exploit immunity to favor persistence. Infection caused by the human immunodeficiency virus type I (HIV-1) is one of the best-studied examples to illustrate this paradox, where APCs act as a double-edged sword throughout the course of infection. Myeloid APCs (such as dendritic cells, monocytes, and macrophages) are not as susceptible to HIV-1 infection as activated CD4⁺ T cells (3). This is likely due to host restriction factors such as SAMHD1 (4, 5) that restrict viral infection and decreases myeloid cell capacity for immune sensing (6), limiting the onset of antiviral responses. However, HIV-1 can exploit myeloid APC biology to reach and infect new target cells through a mechanism that does not rely on the productive infection of myeloid cells. This process was described in vitro at the early nineties by the laboratory of Dr. Ralph Steinman (7), who received the Nobel Prize for discovering dendritic cells. Despite decades of research though, there is no convincing in vivo evidence that demonstrates whether myeloid APCs play a critical role in HIV-1 disease progression.

Upon cellular activation, myeloid APCs can capture and store large numbers of HIV-1 particles (8–10), which are then efficiently transferred to bystander CD4⁺ T cells (7, 11, 12) via cell-to-cell interactions established as part of their immune surveillance routine. Throughout this process of retention and release of virus, HIV-1 exploits a mechanism by which APCs acquire antigens transported by extracellular secreted microvesicles termed exosomes (13). The acquisition of exosomes by activated myeloid APCs contributes to antigen presentation to CD4⁺ T cells (14). This step helps to amplify adaptive immunity without the need for myeloid APCs to be in direct contact with the pathogen (15, 16). Retention of exosomes within intracellular compartments might serve as an antigen depot to control and sustain adaptive immune responses. However, in the case of HIV-1, this internalization route retains infectious particles within protected dynamic compartments (17–19) from where viruses are efficiently transmitted across infectious synapses to susceptible lymphocytes (7, 11, 12).

This particular mode of HIV-1 transmission is known as trans-infection; a route that favors de novo infection of target cells under circumstances where the same dose of cell-free-viruses do not establish productive infection (11). Trans-infection is largely dependent on the expression of the sialic-acid binding I-type lectin receptor Siglec-1 (CD169 or Sialoadhesin) (20–22). Siglec-1 is an interferon inducible receptor constitutively expressed on myeloid cells (23), that is highly upregulated upon myeloid APC exposure to antiviral type I interferons (24, 25). Siglec-1 is a trans-membrane receptor with a long neck that protrudes beyond the glycocalyx of the cell and a terminal V-set domain with the ability to interact with sialylated ligands. While the affinity of Siglec-1 for sialic acid-containing molecules is low, avidity for clusters of sialylated molecules is high (23), allowing for the specific recognition of packaged gangliosides that expose sialyllactose moieties on the viral membrane (26, 27). Likewise, Siglec-1 captures exosomes via recognition of sialylated gangliosides packaged on the microvesicle membrane (21), which assemble and bud from cellular membranes. Murine studies have also confirmed the capacity of Siglec-1 expressed on lymphoid tissues to capture exosomes in vivo (28). Pioneering reports suggested that DC-SIGN, a C-type lectin expressed on immature DCs that patrol peripheral mucosae in search of invading pathogens, could capture HIV-1 early after viral invasion, travel to lymphoid tissues, and establish productive CD4⁺ T cell infection via trans-infection (11). While C-type lectins such as DC-SIGN recognize the viral envelope glycoprotein (11), capture of HIV-1 via Siglec-1 is independent of this interaction (10, 21). Siglec-1 viral uptake largely exceeds the capacity of C-type lectin receptors for HIV-1 capture (21), making this process much more infectious and underscoring novel scenarios within secondary lymphoid tissues in which Siglec-1 trans-infection could fuel viral dissemination.

The molecular pathways governing HIV-1 trans-infection via Siglec-1 on APCs have been described in vitro using both monocyte-derived APCs (20–22) and primary myeloid cells directly isolated from human tissues (25) (Figure 1). Another retrovirus, the murine leukemia virus (MLV), also contains sialylated gangliosides and is captured via Siglec-1 in vitro (29). MLV exploits Siglec-1-mediated trans-infection of permissive lymphocytes to establish infection within secondary lymphoid tissues in mice (30) (Figure 1). However, the in vivo contribution of Siglec-1 to HIV-1 disease progression remains largely unknown.

The lack of available animal models to study HIV-1 infection makes it challenging to investigate the role of Siglec-1 on HIV-1 pathogenesis in vivo. Humanized mouse models susceptible to HIV-1 infection are only established in a few laboratories, and how disease progression in these animals correlates with human pathogenesis needs further investigation. Primate models are also restricted to specific facilities and rely on the use of primate retroviruses that may not directly reflect the biology of HIV-1. Under these circumstances, finding naturally occurring human knockouts could be a good alternative to address the role of key receptors such as Siglec-1 under physiological settings of infection. A deletion in the gene that codes for the HIV-1 co-receptor CCR5, which is needed for acquisition of CCR5-tropic HIV-1, is one of the best-known examples of how a genetic variant alters the phenotype of infection (31). These types of variants have been observed for decades and have also been confirmed by large-scale genomic analysis (32). However, these large-scale analyses have not uncovered novel candidates that might influence HIV-1 disease progression because the available sample sizes are not adequate to assess all possible classes of genetic variation, such as rare and low frequency polymorphisms.

The identification of individuals harboring rare, loss-of-function genetic variants provides an opportunity to study gene function in vivo. Recently, large catalogs of sequenced human genomes have demonstrated that individuals carrying homozygous loss-of-function variants, or natural human knockouts, can provide insight into genetic causes of disease and holds tremendous potential for identifying drug targets (33). However, given their generally low frequency, such variants have gone largely undetected in large-scale genomic analyses. As an alternative strategy to identify genes involved in HIV-1 progression, we conducted a search for individuals lacking the expression of Siglec-1 receptor to study the natural course of HIV-1 infection in the absence of this particular receptor (34). We focused on two well-established cohorts of HIV-1 infected individuals that had been longitudinally followed for decades and had extensive clinical records. We identified two homozygous and almost a 100 heterozygous subjects for a particular stop codon variant in the SIGLEC1 gene. This stop-gain allele is found at highest frequency in individuals of European and South Asian ancestry (1.3%) and is rare or absent in African and East Asian populations (0.5%). Ex vivo experiments confirmed that cells from these individuals were functionally null or partially defective for Siglec-1 expression and, consequently, lost their activity in HIV-1 capture and transmission in vitro. While the lack of Siglec-1 is likely to abrogate trans-infection, the classical HIV-1 infection routes, including cell-free virus infection or cell-to-cell HIV-1 transmission still operate in the absence of Siglec-1, explaining the observation of Siglec-1 null individuals that are HIV-1 infected. However, despite the lack of impact on susceptibility, HIV-1 dissemination and disease progression in infected individuals with null or diminished Siglec-1 expression could be delayed compared with wild type individuals. Nonetheless, we did not observe an effect of Siglec-1 truncation on progression to AIDS.

Several challenges explain the lack of conclusive results in the study of Siglec-1 genetic variants (Figure 2). Power simulations indicate that analysis of a rare variant such as the Siglec-1 allele would require more than 10,000 individuals to detect a relative risk of 5 at P < 0.05 under a recessive model—an effect that would be similar to the beneficial outcome of B*57:01 on HIV-1 control (32, 35–37). This sample size far exceeds even the largest genome-wide studies of HIV-1 progression that comprises ~6,000 patients (38), which does not genotype the Siglec-1 stop variant and cannot be used to impute the presence of this rare allele. Given that the proposed effect requires long-term follow-up off therapy, it is extremely unlikely that a sufficient sample size could be reached to assess the long-term consequences of the Siglec-1 stop variant on HIV-1 disease. Another limitation faced was the lack of seroconversion date for most of the individuals screened; a clinical record that is normally missing in most cohorts of HIV-1 infected individuals. Thus, disease progression was only followed from the date of diagnosis, which may differ between individuals, especially if they are protected by beneficial phenotypes. Moreover, additional clinical data were missing from key individuals, even though we had focused on cohorts with exhaustive follow-up. Indeed, one of the homozygous individuals found had no clinical records for nine years, and information only resumed after antiretroviral treatment initiation, when viral suppression abrogated any potential effect that the Siglec-1 variant might have had on disease progression. Since current clinical guidelines recommend treatment introduction early after HIV-1 diagnosis (39), in the near future this type of analyses will be restricted to retrospective cohorts, which followed old recommendations and started treatment when CD4 counts dropped below a certain threshold, offering a window of opportunity to monitor the natural course of infection. Finally, complexity also arises from the analysis of phenotypes that can be influenced by the infection of several pathogens at the same time. Indeed, one of the homozygous individuals for the rare Siglec-1 allele had a high CD4⁺ T cell count that dramatically dropped when tuberculosis was diagnosed. Lack of Siglec-1 could have had a negative impact on the immune control of the mycobacterial infection, masking any putative beneficial effects caused during HIV-1 progression. Previous studies indicate that Siglec-1 expression on myeloid APCs has a role in combating sialylated bacteria (40, 41). Although sialylation of Mycobacteria has not been documented to our knowledge, direct interaction between Siglec-1 and Mycobacteria might not be required to impact antibacterial immunity. Alternatively, the lack of Siglec-1 on myeloid APCs could compromise antigen capture via exosome or microvesicle transfer and affect the control of the bacterial infection (42–45).

Overall, difficulties and questions faced throughout the study of Siglec-1 null individuals infected with HIV-1 illustrate the major challenges of the field of human knockout genetics applied to infectious diseases. We need to address the biological function of knockout genes of interest in vivo and the effect of a particular variant on health-related phenotypes (46). Variability in the observed phenotypes arises not only from the effect that other genetic variants might have on the gene of interest, but also from the exposure to particular environmental conditions, including the co-occurrence of infections. Animal studies could help to dissect the contribution of these factors by creating the same genetic background and similar environmental conditions in pathogen-free facilities, where co-infections could be experimentally controlled. Working with adequate animal models is, however, complex in the case of HIV-1. An interesting alternative to unambiguously test the potential contribution of myeloid APCs via Siglec-1 to HIV-1 disease progression could be to develop antiviral therapeutic agents against Siglec-1. The identification of Siglec-1 null individuals demonstrates that this protein is dispensable, and its therapeutic blockade is therefore expected not to cause serious side effects. Future work targeting Siglec-1 could provide conclusive evidence of the real contribution of myeloid APC to HIV-1 pathogenesis in vivo. If proven effective, this new family of antiviral agents against HIV-1 could also offer protection against other retroviral infections by mimicking the loss-of-function mutation found in SIGLEC1.