Opinion Cellular Superspreaders: An Epidemiological Perspective on HIV inside the Body

Kristina Talbert-Slagle1,2*, Katherine E. Atkins1, Koon-Kiu Yan3, Ekta Khurana3, Mark Gerstein3, Elizabeth H. Bradley1,2, David Berg2,4, Alison P. Galvani1, Jeffrey P. Townsend1 1 Yale School of Public Health, New Haven, Connecticut, United States of America, 2 Global Health Leadership Institute, Yale University, New Haven, Connecticut, United States of America, 3 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America, 4 Yale University School of Medicine, New Haven, Connecticut, United States of America

Introduction CD4+ T cells may play a similarly critical population, there exists a distribution of role in the establishment and spread of individual reproductive numbers, of which Worldwide, more than 250 people HIV in the genital mucosa after sexual R0 is the mean [14]. In populations of become infected with HIV every hour exposure. homogeneous individuals, the distribution [1], yet an individual’s chance of becom- of individual reproductive numbers will be ing infected after a single sexual exposure, Basic and Individual clustered around the population average the predominant mode of HIV transmis- value of R , and thus, this average value Reproductive Number 0 sion, is often lower than one in 100 [2]. will more accurately predict the likelihood When sexually transmitted HIV-1 infec- To quantify the spread of infectious of from each infected to each tion does occur, it is usually initiated by a disease, epidemiologists use the basic . If R0.1, then an single virus, called the founder strain, reproductive number, R , which describes outbreak is likely to become an , despite the presence of thousands of 0 the average number of secondary infec- and if R0,1, then an outbreak will not genetically diverse viral strains in the tions that arise from one infected individ- spread beyond a few initially infected transmitting partner [3]. Here we review ual in an otherwise totally susceptible individuals [15,16]. evidence from molecular biology and population [15]. The basic reproductive In heterogeneous populations, however, virology suggesting that heterogeneity number can be approximated as the the population average value of R0 is less among CD4+ T cells could yield wide product of the following: (1) the average predictive of transmission dynamics [14]. variation in the capability of individual number of susceptible individuals contact- For example, in populations with highly cells to become infected and transmit HIV ed by an infected individual during the right-skewed distributions of individual to other cells. Using an epidemiological (the ‘‘number of con- reproductive numbers, most individuals framework, we suggest that such hetero- tacts’’) and (2) the average probability that infect few, if any, others, but a few geneity among CD4+ T cells in the genital a susceptible individual will become in- individuals infect many others. In such mucosa could help explain the low infec- fected by a single infected individual populations, there is a high probability tion-to-exposure ratio and selection of the during its infectious period (the ‘‘shedding that a will not be founder strain after sexual exposure to potential’’). Thus, sustained in the population and will HIV. instead go extinct [14]. In some cases, During sexual transmission, founder however, those rare individuals in the tail R &Number of contacts viral strains preferentially infect CD4+ T 0 of the distribution with a much higher- cells using the CCR5 coreceptor [4,5]. At |Shedding potential: than-average individual reproductive the time of initial exposure to HIV, these number while they are infected, known CD4+ T cells exhibit baseline heterogene- as ‘‘superspreaders’’ [15], can have a ity due to stochasticity in cellular gene The number of secondary significant impact on whether an outbreak expression [6] and dynamic variation in caused by a specific individual throughout becomes an epidemic or goes extinct. immunological status (activated, resting, the time that the individual is infectious is Epidemiological outbreak investigations, etc.) [7]. In addition, because CD4+ T called the ‘‘individual reproductive num- which track the spread of disease by a cells are mobile, they are heterogeneously ber’’ [14]. For any disease within a given technique called , have distributed in the genital mucosa, with varying degrees of clustering and contact Citation: Talbert-Slagle K, Atkins KE, Yan K-K, Khurana E, Gerstein M, et al. (2014) Cellular Superspreaders: An [8–11]. In other contexts, it is well-known Epidemiological Perspective on HIV Infection inside the Body. PLoS Pathog 10(5): e1004092. doi:10.1371/ that heterogeneity among isogeneic cells journal.ppat.1004092 inside the body can affect many cellular Editor: Glenn F. Rall, The Fox Chase Cancer Center, United States of America behaviors and outcomes, including infec- Published May 8, 2014 tion dynamics [12,13]. Copyright: ß 2014 Talbert-Slagle et al. This is an open-access article distributed under the terms of the Epidemiological analyses of disease Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any outbreaks among people indicate that medium, provided the original author and source are credited. heterogeneity in the ability of individuals Funding: KTS was supported by a Ruth L. Kirschstein National Research Service Award (http://grants.nih.gov/ in a population to spread disease can have training/nrsa.htm). The funders had no role in study design, data collection and analysis, decision to publish, or a significant impact on whether a local preparation of the manuscript. outbreak becomes an epidemic [14]. Competing Interests: The authors have declared that no competing interests exist. Heterogeneity among a population of * E-mail: [email protected]

PLOS | www.plospathogens.org 1 May 2014 | Volume 10 | Issue 5 | e1004092 identified the existence of superspreaders among CD4+ T cells in the genital mucosa is not close enough to others to transmit in many well-known infectious disease of a single individual could generate a virus by direct contact [8]. Thus, hetero- outbreaks, including , mea- skewed distribution in the individual geneity in cell distribution and clustering sles, , , and severe acute cellular reproductive number, or ICRN, inside the body could generate wide respiratory syndrome (SARS) [14,17,18]. in the context of HIV infection. Here we variation in the efficiency of virus trans- These rare individuals often make a review evidence for heterogeneity among mission from cell to cell and in ICRN. significant, sometimes deciding, contribu- CD4+ T cells that could lead to wide Transmission of virus from an infected tion to the dynamics of disease spread variation in ICRN and possibly give rise to to a susceptible cell also depends on a cell’s (Table 1). cellular superspreaders. permissivity to productive infection. The During the 2003 SARS outbreak in level of surface expression of CD4 and Singapore, for example, the majority of Number of Contacts CCR5 (the predominant coreceptor uti- individuals who became infected spread lized during acute infection [32]) varies the virus either to no one else or to only CD4+ T cells exhibit considerable widely among CD4+ T cells [25,33], even one other [14]. Five infected individuals, heterogeneity in activation status (e.g., in a single individual [34], and affects however, were superspreaders, each in- resting or activated) and expression of cellular permissivity to HIV [35,36]. fecting at least 20 others (Figure 1) [19]. In surface molecules important for HIV Indeed, low expression of CD4 or CCR5 this example, R0, which is an average infection (including the HIV coreceptors can completely inhibit infection of CD4+ population value, did not adequately CCR5 and CXCR4) in human penile T cells by certain viral strains [37]. A describe the dynamics of SARS because [21], foreskin [22,23], cervical [24,25], recent multiparameter analysis of HIV it did not capture the heterogeneity among and rectal [26] tissue. In addition, various entry efficiency at the level of single cells individuals in their ability to spread disease studies that stain for CD4+ T cells in indicated large cell-to-cell variation in or the key contribution made by super- uninfected genital mucosal tissue, such as expression of CD4, CCR5, and the spreaders to establishment and spread of cervical tissue [27] and human foreskin coreceptor CXCR4, which subsequently the virus [14]. [22], indicate that T cells vary in their influenced permissivity of individual cells spatial distribution and the extent to which to HIV binding and entry [38]. In Individual Cellular they form clusters. Cell density and spatial addition, CD4+ T cells isolated from Reproductive Number arrangement have been identified as rectal and cervical tissue exhibit consider- important sources of heterogeneity among able heterogeneity in expression of the For a population of HIV-infected cells cells that can affect virus spread in vitro surface integrin a4b7, which can specifi- inside the body, the basic reproductive [8,28–30]. Indeed, imaging studies explor- cally bind the V2 loop of the HIV number, R0 (a population average), has ing the dynamics of virus spread in a envelope protein gp120 and may improve been quantified [20]. As with individual model of sexual transmission to female cell-to-cell spread by activating other cell- humans inside a population, however, nonhuman primates indicate that virus surface molecules in the viral synapse [39]. empirical evidence indicates that individ- spreads unevenly among clusters of cells in Experiments in vitro indicate that HIV ual human cells inside a population are the endocervix [9]. Cells in these clusters preferentially infects cells expressing high also heterogeneous [12], varying in their tend to be in close proximity, and if cell-to- levels of CCR5 and that infection can be contact with one another, their ability to cell transmission is far more efficient than further enhanced by high levels of surface become infected (permissivity), and also in cell-free transmission, as some studies a4b7 expression in some individuals whether and to what extent they transmit suggest [31], then a cell that is physically [40,25]. Heterogeneity among CD4+ T infectious virus to other cells during their touching its neighbors could generate cells in expression of specific cell surface infectious period. Such heterogeneity more secondary infections than a cell that receptors can thus affect permissivity and

Table 1. Superspreading events during infectious disease outbreaks.

a b Disease Location (year) (R0) SSE References

EBOLA Congo (1995) 1.83 21+, 28–38 [85,86] Greenland (1951) 16 250 [15,87] US (1985) 16 69,84 [15,88] Canada (2011) 16 678 [15,17] PNEUMONIC PLAGUE China (1946) 1.3 32 [89,90] SARS (2003) 3 187 [91,92] Vietnam (2003) 3 20 [91,93] Singapore (2003) 1.6 12,21,23,23,40+ [14,19] Canada (2003) 3 19,12–24 [91,94] SMALLPOX Yugoslavia (1975) 5.5 38 [95,96]

a R0: The average number of secondary cases caused by an infected individual during the outbreak; here, R0 is reported either for a specific outbreak, when available, or as a measure calculated based on multiple past outbreaks. bSSE: Superspreading events—number of infections caused by a single individual during an outbreak; number of infections caused by multiple superspreading events during the same outbreak are separated by commas. This table is adapted from the supplementary material from reference [14]. doi:10.1371/journal.ppat.1004092.t001

PLOS Pathogens | www.plospathogens.org 2 May 2014 | Volume 10 | Issue 5 | e1004092 gene expression and likely also subsequent virus shedding potential from any given cell. Within the population of CD4+ T cells in the genital mucosa, therefore, a wide range of ICRN may exist due to stochastic and/or infection-driven variations in viral gene expression and viral particle produc- tion, arising from potentially wide hetero- geneity in virus shedding potential among infected cells.

Cellular Superspreaders Experiments in a nonhuman primate model of HIV infection have demonstrat- ed that the vast majority of CD4+ T cells Figure 1. Contact tracing of SARS in Singapore showed that most people (gray circles) transmitted the virus to very few others, while a few individuals acted as ‘‘super- in the genital mucosa of a healthy, spreaders,’’ infecting many more people than average. Patient numbers corresponding to uninfected individual are resting cells, those individuals who were identified as superspreaders are shown. All cases trace back to patient which outnumber activated cells 70:1 1 [18]. (Figure 2A) [9]. Activated CD4+ T cells doi:10.1371/journal.ppat.1004092.g001 express higher levels of CCR5 [33] and a4b7 [40] than do resting cells. In cell-to-cell transfer of HIV infection in the Variation in virus gene expression at the addition, experiments in nonhuman pri- genital mucosa. level of individual cells has been demon- mates indicate that infected, activated cells Permissivity of cells to productive HIV strated in vivo in a mouse model of contain five times more viral RNA, release infection can also be affected by intracel- cytomegalovirus infection [45], and het- 10-fold more viral particles to their lular proteins called restriction factors, erogeneity among individual cells in the surrounding environment, and tend to which block the progress of HIV through production of virus particles, or virus form larger cell clusters than resting cells the cell [41]. Expression of some cellular shedding, has been shown through analy- [9]. Notably, although the nonhuman restriction factors has been shown to vary sis of cells isolated from simian immuno- primate model has long been used to among different human populations [42] deficiency virus (SIV)–infected nonhuman study many facets of HIV infection [53], and even between different types of CD4+ primates, in which rare activated CD4+ T the virus used in these experiments, SIV, T cells within the same individual [43]. cells were shown to individually release expresses a protein that allows it to Notably, an intracellular restriction large quantities of virus [10]. Here we productively infect resting CD4+ T cells. factor known as SAMHD1 (sterile alpha focus on variability in gene expression and In contrast, HIV-1 does not express this motif [SAM] and histidine/aspartic acid its potential impact on an individual cell’s protein and is thus unable to generate [HD] domain-containing protein 1) blocks capability to release infectious virus as a productive infection in resting CD4+ T reverse transcription in resting—but not possible source of heterogeneity in shed- cells [54]. Resting human CD4+ T cells in activated—CD4+ T cells, inhibiting HIV ding potential. the human genital mucosa are therefore replication. These findings help to explain Stochasticity in cellular gene expression even less likely to be able to produce and the inability of resting CD4+ T cells to is a common phenomenon [6] and has spread HIV than are resting CD4+ T cells produce infectious virus [44]. HIV com- also been observed in viral gene expres- in a nonhuman primate model. bats the effects of some cellular restriction sion, including HIV. In populations of Together, these data suggest that upon factors with its own viral proteins, but the genetically identical cells infected with infection with HIV the majority of CD4+ success of these proteins in overcoming HIV, viral genes tended to be expressed T cells in the genital mucosa would spread cellular resistance and facilitating virus at either high or low levels but were also the virus to few if any others and that only production depends on their quantity rarely expressed at intermediate levels, rare cells would have the capacity to within the cell, which can also vary varying from cell to cell [46]. release large quantities of HIV to their depending on viral gene expression levels The site of virus integration also affects nearest neighbors. These rare cells may be [41]. viral gene expression. HIV viral DNA activated CD4+ T cells, which have been Together, these data suggest that preferentially integrates at sites of active described as ‘‘amplifiers’’ that can cause among CD4+ T cells in the genital cellular gene expression although not at any additional cells to become infected with mucosa, significant heterogeneity may specific site or in any specific gene [47–49]. HIV [10,55]. Such rare cells may exhibit a exist in the number of contacts that Some viral integration events occur at sites specific set of traits that facilitate estab- become infected by any given infected cell of much higher gene expression than others lishment of HIV inside the body. For even within the same host, potentially [47], and gene expression can vary signif- example, experiments done in vitro indi- leading to a skewed distribution of ICRN. icantly depending on the site of integration, cate that CD4+ Th17 cells, which express even in genetically identical cells [50]. Since high levels of CCR5 as well as the Shedding Potential the majority of HIV-infected cells in lymph chemokine receptor CCR6 and the inte- nodes and peripheral blood contain a single grin a4b7, are preferentially targeted Release of infectious viral particles from virus [51], while cells in splenic tissue during early infection, and analysis of an infected CD4+ T cell, or shedding contain one to eight integrated proviruses samples from female sex workers who are potential, can be influenced by many (mean 3.2) [52], viral integration site can be infected with HIV indicate that CD4+ factors in both the cell and the virus. an important factor in determining viral Th17 cells are selectively depleted from

PLOS Pathogens | www.plospathogens.org 3 May 2014 | Volume 10 | Issue 5 | e1004092 Figure 2. Heterogeneity among CD4+ T cells in the genital mucosa and the HIV founder strain. (A) The majority of CD4+ T cells in the female genital mucosa of an uninfected individual are resting cells; rare cells are activated. (B) Most virus particles remain trapped in the mucus that coats the cervical epithelium though a few can enter through microabrasions. Resting CD4+ T cells can become infected with HIV but do not produce infectious virus. Infection of activated CD4+ T cells, which tend to form clusters, have higher levels of gene expression than resting cells, and produce infectious virus, may be the that establishes the HIV founder strain after sexual transmission. ICRN: individual cellular reproductive number. Based on data from references [3] and [10]. Images of cervical epithelium by OpenStax College [CC-BY-3.0 (http:// creativecommons.org/licenses/by/3.0)] via Wikimedia Commons. doi:10.1371/journal.ppat.1004092.g002 the cervix during HIV infection [25,56– reproductive number for most individuals. infected, which has been shown to occur 58]. In addition, given that the majority of As shown in Figure 1, infection of a single within days after infection in vitro [60,61] HIV infections begin with a single found- superspreading individual (labeled as ‘‘1’’) but has yet to be confirmed in vivo, a ing strain [59] and most infected cells in triggered the SARS outbreak in Singapore ‘‘local outbreak’’ inside the body would go peripheral blood and lymph node tissue in 2003. extinct. In this case, infection of most cells contain a single copy of HIV DNA immediately after sexual exposure would (although these may or may not be Implications of Cellular not lead to sustained infection, which representative of HIV integration in mu- Superspreaders in HIV Infection could explain the very low infection-to- cosal lymphatic cells) [51], infection of a exposure ratio for sexual exposure to HIV. single cell has the potential to establish A highly skewed distribution of individ- Second, such short-lived ‘‘local out- HIV infection inside the genital mucosa ual reproductive numbers in a population, breaks’’ of HIV within an individual could after a given exposure. Infection of a rare in which most individuals infect few if any still yield a low level of virus production, CD4+ T cell with very high ICRN could others but a tiny minority are super- even if the initial outbreak of HIV thus be the superspreading event that both spreaders, has two important implications infection ultimately goes extinct. Among establishes HIV infection in the genital when applied to CD4+ T cells in the a group of nonhuman primates exposed to mucosa and selects a single founder strain genital mucosa. First, since the majority of a low physiological dose of SIV, some (Figure 2B). the cells would have a low ICRN, most, if animals experienced initial low levels of Such a founding superspreader infec- not all, of the viral strains that successfully viral replication and immune response tion event would parallel, for example, overcome physiological barriers during without ever proceeding to full infection the dynamics that governed the SARS exposure are likely to infect cells that have or seroconversion, a phenomenon called outbreak in Singapore, giving rise to an ICRN less than one. Provided that occult infection [62–64]. In addition, some an epidemic despite a low individual none of these cells becomes latently HIV-exposed, seronegative humans who

PLOS Pathogens | www.plospathogens.org 4 May 2014 | Volume 10 | Issue 5 | e1004092 continue to engage in high-risk sexual cells in the genital mucosa gives rise to tion. Several recent studies suggest that behavior exhibit immunological markers cellular superspreaders that may drive Th17 cells, a subset of CD4+ T cells, are that indicate a prior immune response to establishment of HIV infection inside the preferentially infected by early viral strains HIV infection even though they remain genital mucosa after sexual transmission. and selectively depleted from the cervix seronegative, supporting the idea that local The definition of R0 provided here during HIV infection [58,25]. We are not infections may have occurred in these implicitly integrates transmission over the aware of in vivo data that explicitly support patients [65–67]. Finally, analysis of the time that an index cell was infected, the existence of cellular superspreaders; unsuccessful HIV vaccine STEP trial meaning that we have incorporated dura- nevertheless, the data reviewed here suggest suggested that a large portion of the tion of infectiousness into our overall their existence, warranting further empiri- exposed individuals may have experienced definition of R0. Thus, a CD4+ T cell cal research. Identification and targeting of occult infection, implying that this phe- could theoretically become a supersprea- cells most likely to become superspreaders nomenon could be more widespread than der either by spreading a large amount of could facilitate the development of preex- previously suspected [68]. There is, how- infectious virus to other cells in a short posure or immediate postexposure thera- ever, no direct experimental evidence for amount of time, or by spreading a smaller pies that could prevent a local outbreak of occult infection in humans. amount of virus to other cells for a HIV inside the genital mucosa from If infection of a rare superspreader comparatively longer period of time, or becoming a within-host epidemic that CD4+ T cell establishes the founder strain, through some combination of the two. spreads throughout the body [9,83]. then the few features of founder viral Though any of these mechanisms are In this review, we have applied epide- strains that have been observed might in possible in early infection [79], studies in miological concepts of disease spread fact confer a selective advantage during nonhuman primates suggest that establish- specifically to explore unsolved questions early infection. These features include ment of infection in the genital mucosa regarding establishment of the HIV found- shorter envelope glycoproteins with fewer typically occurs within 3–7 days after er strain and the low infection-to-exposure N-linked glycosylation sites [69,70] as well male-to-female sexual exposure [62,80]. ratio of infection after sexual transmission. as preferential infection of CD4+ T cells The lifespan of a productively infected We suggest that these concepts may also expressing high levels of CCR5. Some CD4+ T cell is on average 2.2 days [81]. be applied more broadly to explain the founder viral strains have also exhibited Thus, in the specific case of HIV infection documented existence of HIV founder high affinity for the a4b7 integrin receptor in the genital mucosal tissue, if infection strains after transmission via injection drug [71,72] though this has not been univer- becomes established via a superspreading use and from mother to child [59] since sally observed [73,5]. The founder virus, event, it is likely to occur within the first CD4+ T cells in the blood of healthy, which more closely resembles the ancestral few days of exposure and be driven by a founder strain than it does the predomi- uninfected individuals are also heteroge- relatively short-lived, productively infected neous, with only a very small subset nant strain in the transmitting partner cell that generates a much higher-than- [74], may be selected by its ability to infect exhibiting an activated or replicating average number of secondary infections a subtype of CD4+ T cell: a cellular phenotype [84]. due to a high shedding rate, or a high superspreader. This specific efficiency Since heterogeneity among cells has contact rate, or both. Notably, as in other could explain why HIV founder strains been acknowledged as an important factor superspreading events, establishment of have not shown a consistent infectivity in a variety of cellular processes, including HIV by infection of a cellular super- advantage in CD4+ T cells over strains certain viral infections [12], we suggest spreader could occur even if the basic from chronic infection in vitro [5,75,76], that the epidemiological framework de- reproductive number for the entire popu- as these experiments likely do not fully scribed here may also be applicable to the lation of susceptible cells is low. replicate the cellular heterogeneity of the establishment and spread of other cellular If cellular HIV superspreaders do exist, in vivo environment [32,77,78], where the diseases inside the body, including not and if they are the cellular culprits driving founder viruses might have an advantage only infections but perhaps also certain in infecting the rare superspreader cells. the establishment of HIV infection inside cancers. The impact of cellular heteroge- the body, then the most successful strategy neity may be particularly profound if the for preventing the infection from becoming Conclusions distribution of individual cellular repro- established in the body is to block or ductive numbers is highly skewed, yielding Here we have applied two key concepts remove these cells before or shortly after cellular superspreaders. from : R0, which is the infection in order to drive a local, within- approximated product of number of host outbreak to extinction [13,82]. Such Acknowledgments contacts and shedding potential through- cells may have specific traits, such as high out the infectious period, and individual expression of surface receptors including We thank Pradeep Uchil, Walther Mothes, and reproductive number, to suggest that a CCR5 and possibly also a4b7, that allow Daniel DiMaio for helpful discussions and skewed distribution of individual cellular them to be identified and targeted by novel Maggie Zhou for contributions to the manu- reproductive number among CD4+ T therapies to prevent establishment of infec- script revision.

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