Discussion. Part 2
In order to better characterize the relationship between P. aeruginosa and mammalian cells during infection, we examined the effect of this organism and its fractions on 3 hydrolases present in mouse monocytes. Experiments with both living and intact dead P. aeruginosa in vitro and in vivo indicated that beta glucuronidase and alkaline phosphatase were affected by intact bacteria. Although the response of beta glucuronidase varied in certain experiments, the results in table 8 clearly show inhibi tion of activity in the presence of living extracell ular organisms. The response of alkaline phosphatase suggested that both stimulation and inhibition of this enzyme occurred during infection. Viable organisms incubated with monocytes apparently did not destroy the cell’s ability to produce or activate alkaline phosphatase, despite the fact that previous manometric studies by Berk et al (1960a) had indicated that resting cells of P. aeruginosa metabolized monocytes. When monocytes were infected in vivo (table 10), acid phosphatase was depressed, while alkaline phosphatase was stimulated. This was reminiscent of the increased lactate production found with infected cells (Berk et aI, 1960a).
The response of monocytes allowed to ingest dead P. aeruginosa in vivo closely paralleled that obtained when live bacteria were used. In addition to these in vivo studies, similar experiments performed in vitro (where little or no ingestion occurred) yielded basically similar results. The results in table 3 indicate that trace amounts of piromen exerted some effect on all 3 enzymes. Beta glucuronidase appeared most susceptible to piromen. Depression of enzyme activity was not directly proportional to piromen concentration. This suggests that inhibition may be a reflection of some cell surface phenomenon, especially since it has already been demonstrated by Kerby (1952) that piromen disrupts the cell structure of leucocytes. Experiments utilizing particles of P. aeruginosa sedimented at 35,000 ref (Alexander, 1956) yielded results very similar to those with piromen. The particles were more toxic (on a nitrogen basis) for hydrolase than for succinoxidase activity. Similar studies with toxic particles from hemolytic streptococci have been reported by Roberson et al (1960). An analysis of the over-all response of P. aeruginosa-infected monocytes indicates that lactate production and alkaline phosphatase activity increase, while succinoxidase and acid phosphatase activities decrease. Cytochrome oxidase is unaffected, while the effect on beta glucuronidase is variable. It appears that certain enzymes are more susceptible to certain microorganisms than to others and that some variation may be anticipated, depending on experimental conditions. In general, the effect of either piromen or the subcellular particles seems adequate to account for the toxicity of intact P. aeruginosa. The outstanding difference between these lies in the response of alkaline phosphatase. One may suspect, however, that depression in acid phosphatase may be compensated by a corresponding increase in alkaline phosphatase.
DISCUSSION
Previous manometric studies by Berk and Nelson (1960) indicated that succinoxidase activity of mouse monocytes was depressed by a few micrograms of a Pseudomonas polysaccharide (piromen) which contained traces of a lipidnucleic acid moeity. Cytochrome oxidase was relatively unaffected. Further studies (unpublished) indicated that a soluble and a particulate component of disrupted P. aeruginosa inhibited succinoxidase activity of mouse monocytes to a greater extent than did intact bacteria. Similar inhibitory effects on succinoxidase activity of monocytes or spleen and liver homogenates could not be demonstrated with commercially available bacterial lipopolysaccharides (unpublished results). Whereas previous studies (Berk et al, 1960b) indicated that the respiratory enzymes of mouse monocytes were relatively weak, with large numbers of cells required for activity, the studies reported here indicate that these cells are rich in nonoxidative enzymes. Acid phosphatase, alkaline phosphatase and beta glucuronidase were all present in mouse monocytes. In addition, amylase and arylsulfatase have also been found in these cells (unpublished data). Several. investigators (Cohn and Hirsch, 1960a, b; Cram and Rossiter, 1949; Fishman, 1955; Haight and Rossiter, 1946; Roche, 1931) have also shown phosphatases and beta glucuronidase in circulating leucocytes and rabbit polymorphonuclear cells. Using human cells, Haight and Rossiter (1946) found alkaline phosphatase chiefly in polymorphonuclear leucocytes and acid phosphatase mainly in lymphocytes. Roche (1931) demonstrated alkaline phosphatase activity in both granulocytes and lymphocytes from the buffy coat of rabbit and guinea pig blood. Recently, Cohn and Hirsch (1960a, b) reported the presence of hydrolases in rabbit polymorphonuclear leucocytes.
The study of these mouse enzymes was aided by the fact that fewer cells were required than for oxidative studies. Furthermore, despite the fact that some substances stimulated enzyme activity, the enzymes were both stable and active without the use of cofactor supplements. On the basis of cell numbers, beta glucuronidase was especially active in comparison to the 2 phosphatases or to succinoxidase. Activity could be detected with as little as 5 X 104 cells per ml, whereas studies with phosphatases and oxidases required 5 X 106 and 5 X 108 cells per ml, respectively. Attempts to increase the rate of beta glucuronidase activity by using possible activators yielded a variety of results. The influence of bovine serum albumin and other substances on this enzyme for other mammalian tissue preparations has been reported (Bernfeld et al, 1954). Heparin, routinely used to prevent clumping of cells, had no appreciable effect on beta glucuronidase activity in monocytes, though Becker and Friedenwald (1949) found it inhibitory to this enzyme in liver preparations. Unlike bovine serum albumin, sodium chloride and hyaluronic acid, trace amounts of citrate did not stimulate beta glucuronidase. Other investigators (Karunairatnam and Levvy, 1949) have used mouse liver preparations and also found this to be true.
Studies on Iviechanisivis of Cellular Immunity. Part 3
Substrate concentration.-The activity of all enzymes increased as substrate concentrations were raised from 2 to 102 JLM per ml. No substrate inhibition occurred within the limits measured.
Cofactor studies.-The presence of various substances such as magnesium ions, citrate, hyaluronic acid and bovine serum albumin had some effect on the various enzyme systems. Magnesium ions were stimulatory for both phosphatases with maximum stimulation occurring at 3 X 10-5 M:. Beta glucuronidase activity was stimulated by hyaluronic acid (0.008 to 0.04 mg per ml), sodium chloride (0.2 mg per ml) and bovine serum albumin (0.005 to 0.05 mg per ml). Heparin (0.06 to 50 mg per ml) had no effect, while citrate (0.05 to 1.0 mg per ml) was slightly inhibitory.
Experiments with piromen. t- The results in table 3 indicate that both acid phosphatase and beta glucuronidase were inhibited by trace amounts of piromen. Beta glucuronidase activity was depressed 58% by 8.2 JLg piromen per ml and acid phosphatase was depressed 35% by 7.5 JLg. To a slight degree alkaline phosphatase was both stimulated and inhibited.
Experiments with lipopolysaccharides. -The results in tables 4 and 5 indicate that the lipopolysaccharides of several different organisms exerted no appreciable effect on the 3 hydrolases, even though quantities up to 50 JLg per ml were used. Some depression of acid phosphatase activity occurred with highly purified material, but otherwise, when slight stimulation or inhibition was noted, repetitive experiments did not confirm the results.
Experiments with bacterial particles. A particulate fraction prepared from disrupted P. aeruginosa was found to be inhibitory to each enzyme . Alkaline phosphatase and beta glucuronidase were depressed 30% or more by 38 JLg N per ml, while acid phosphatase was depressed 15% by 17.5 JLg N per ml. In vitro experiments with P. aeruginosa.
Acid phosphatase was depressed, alkaline phosphatase was stimulated, and beta glucuronidase was unaffected. Since P. aeruginosa contains neither alkaline phosphatase nor beta glucuronidase, it was possible to carry out similar experiments with live organisms. Alkaline phosphatase was also stimulated when monocytes were incubated with live pseudomonads. The stimulatory response was partially reversed when the concentration of bacteria was increased. Beta glucuronidase was inhibited only by living P. aeruginosa.
In vivo experiments with P. aeruginosa.- When mouse monocytes were allowed to ingest dead P. aeruginosa in vivo, the results obtained were nearly identical to those of previous experiments where no phagocytosis occurred. The results in table 9 indicate that beta glucuronidase activity was depressed. The results of experiments in which monocytes were infected in vivo with live organisms can be found in table 10. The pattern with acid and alkaline phosphatase remained the same as in other experiments. However, the response of beta glucuronidase in infected monocytes was variable.
Studies on Iviechanisivis of Cellular Immunity. Part 2
Acid and alkaline phosphatases were assayed by the method of Bessey et al (1946) with pnitrophenyl phosphate as substrate. The resultant p-nitrophenol was quantitatively determined in cell-free supernatants at pH 10 and 420 mu, Alkaline phosphatase was measured in 0.1 M tris buffer, pH 9.1, and acid phosphatase at pH 4.3 and 0.1 M acetate buffer. The level of substrates in reaction mixtures was 5 to 10 ttM per ml. Two ttM of magnesium chloride were added to acid and alkaline phosphatase reaction mixtures whenever in vivo phagocytosis was omitted. Substrates were obtained from the California Corporation for Biochemical Research; p-nitrophenol was obtained from the Nutritional Biochemical Corporation. Nitrogen.-The method of Wong (1923) was used for nitrogen analysis of bacterial particles. Supplements.-Piromen was obtained from Travenol Laboratories, Inc., Morton Grove, Illinois; lipopolysaccharides from Dr. Arthur Johnson, University of Michigan Medical School, and from Difco Laboratories; and polymyxin B from Burroughs Wellcome.
RESULTS
Enzymes.. These studies were extended to determine the site of enzyme activities in sonically disrupted cells. All 3 enzymes were present in the soluble fraction but not in the cellular debris and particulate fractions obtained at 35,000 ref. All enzymes showed greatest activity within the first 12 minutes after addition of substrate. As the time of incubation increased, the rate of hydrolysis decreased.
Effect of pH.–Beta glucuronidase and acid phosphatase activities were noted from pH 3.5 to 5.2. Alkaline phosphatase was active within a narrower range. The optimal pH for beta glucuronidase was 3.9 and for acid and alkaline phosphatases 4.3 and 9.1, respectively. Temperature studies.-Beta glucuronidase activity was obtained at all temperatures between 4 and 56 C with maximum activity at 37 C. Activity was slight at 4 C, while temperatures above 37 C proved suboptimal. The results differed with the phosphatases in that they were inactive at 4 C and temperatures above 37 C did not affect them. Acid phosphatase values at 45 and 56 C were slightly higher than values obtained at 37 C, while alkaline phosphatase activity was approximately the same at all 3 temperatures.
Cell concentration.- beta glucuronidase activity can be detected in preparations containing as few as 5 X 104 cells per ml. As the cell concentration was increased enzymatic activity also increased. Similar studies with the phosphatases indicated that activity was not detectable unless 5 X 106 cells per ml were used. In every case, acid phosphatase was more active than alkaline phosphatase.
STUDIES ON IVIECHANISIVIS OF CELLULAR IMMUNITY
The relationship between microorganism and host cell during development of an infectious process has never been clearly defined. In order to fully understand the effect of a microbial pathogen on a mammalian cell, it is important to know how each affects the enzymatic machinery of the other. Recently there have been some investigations of the biochemical aspects of host-parasite interactions (Berk and Nelson, 1960; Berk et al, 1960a, b; Kun and Miller, 1948; Puziss and Wright, 1959; Suter, 1956). Studies in our laboratory with a mouse monocyte-Pseudomonas aeruginosa system have indicated that host cell metabolism can be disturbed radically during contact with an infectious agent. For example, succinoxidase activity of in vivo infected mouse phagocytes was depressed, while lactate production from hexoses was markedly stimulated (Berk et al, 1960a, b). It is the purpose of this paper to describe 3 hydrolases in mouse mononuclear cells and to report the effect of P. aeruginosa on the activity of these enzymes.
METHODS
The procedures for obtaining exudates and maintaining the streptomycin-resistant strain of P. aeruginosa were essentially those previously reported (Berk and Nelson, 1960; Nelson and Becker, 1959). Warm mineral oil (0.5 ml per mouse) was used to induce peritoneal exudates in Received for publication May 13, 1961. * This investigation was supported by research grant E-2298 from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. 8 to 10-week-old female mice (Webster strain) 18 hours before harvesting of cells. The pooled monocytes from 40 mice were washed twice with saline by centrifuging for 10 minutes at 500 ref. Hemocytometer counts indicated that 1 ml contained approximately 5 X 108 packed cells. Most experiments employed intact cells, but when disrupted cells were required homogenates of equivalent numbers were prepared by sonic treatment in a 9 kc per second Raytheon magnetostrictive apparatus or by disruption at 20,000 psi in a French pressure cell (Aminco). Cellular debris was centrifuged off in the cold at 600 ref and the resultant supernatant was recentrifuged at 30,000 to 35,000 ref for 20 to 30 minutes. In some cases a minute amount of light yellow-green sediment was obtained at this speed. In experiments with infected mice, 109 to 10ro washed P. aeruginosa were administered intraperitoneally 15 to 60 minutes before autopsy. Monocytes were washed 2 to 3 times with heparinized saline containing 1000 to 4000 units of polymyxin per ml to remove and destroy extracellular bacteria. In some experiments 1000 units of polymyxin per ml were also incorporated into reaction mixtures. Similar procedures were used when monocytes were allowed to ingest dead microorganisms in vivo. In experiments employing toxins or bacteria in vitro, monocytes were pre-incubated with these supplements for 2 minutes prior to substrate addition. The pH of all supplements was adjusted to the optimal pH for each enzyme. Particles were obtained by disrupting the bacteria in a French pressure cell at 20,000 psi, centrifuging at 2000 ref to remove cellular debris, and concentrating at 35,000 ref. Assay.-Beta glucuronidase was assayed by the method of Dodgson et al (1953) with phenolphthalein glucuronide as substrate. Into test tubes were pipetted 20 to 50 ,aM of substrate, 2 ml of 0.1 M acetate buffer, pH 3.9, and 0.5 ml of a monocyte suspension in acetate buffer. The heparin concentration of the reaction mixtures was 20 units per mI. The reaction mixtures were incubated at 37 C for 30 to 60 minutes. At the end of the incubation period, 1 ml of 10% trichloroacetic acid was added and the resultant super innatant was assayed after centrifugation. The amount of phenolphthalein liberated was determined by its color intensity at pH 10.5 and 540 m«.
Influence of Host Genetic Variation on Susceptibility to HIV Type 1 Infection
As a prelude to the discussion of host genetic determinants of susceptibility to HIV‐1 infection, we briefly enumerate the nongenetic factors involved and the major issues that arise in designing and analyzing research on genetic variation. Subsequent sections emphasize the importance of evaluating genetically mediated predisposition to infection in the context of genetic influences on clinical responses in individuals who are already infected. Similarities and differences between these 2 types of effects may help distinguish between those factors more involved in initial viral penetration, those more involved in long‐term host adaptation to established infection, and those equally important to both processes.
Nongenetic Influences on Transmission and Acquisition
Biological factors.Viral and host biological characteristics are strong predictors of both transmission and acquisition of HIV‐1. The infectivity of HIV‐1 in a potential “donor” may differ from that in another because of differences in virus subtypes and the set of virus quasi species that are more or less well adapted to the biological individuality of that donor. The envelope glycoprotein of HIV‐1 is one major factor governing its in vitro tropism for certain host cell types, and differences in such properties as size, shape, and net charge of certain envelope motifs could alter the capacity of the virus to penetrate host cells. The duration of infection and the effectiveness of the immune response in the donor further dictate qualitative and quantitative characteristics of virus replication and the capacity to spread to susceptible “recipients.” In general, virus load during the acute and latter stages of infection is higher than during the long middle (“latent”) period, and higher donor virus load at those stages presumably increases the likelihood of transmission to a naive host. Another critical factor is the condition of immune activation in both donors and recipients, especially in areas of close contact; the absence of circumcision, ulceration, and other causes of mucosal disruption presumably increases access to or activates cells targeted by HIV‐1. The age of the recipient may be a surrogate for maturation or senescence in the host cellular immune processes involved in defending against viral penetration and integration. Infection with herpes simplex viruses and, perhaps, other viral and bacterial agents may facilitate propagation of HIV‐1 by activating cells or otherwise promoting virus replication and shedding. Undoubtedly, other recipient immune defense mechanisms that can modulate the risk of infection are still undiscovered.
Valganciclovir and Human Herpesvirus–8
The possibility that ganciclovir or valganciclovir might inhibit HHV‐8 replication and prevent development of Kaposi sarcoma in HIV‐infected patients was suggested by the observation that patients who received 4.5 g daily of orally administered ganciclovir to prevent development of CMV retinitis in the eye without an ocular ganciclovir implant also had a lower incidence of Kaposi sarcoma in a large clinical trial. This suggested the intriguing possibility that replicating HHV‐8 expressed lytic proteins or stimulated cellular cytokines, which contributed to the development of Kaposi sarcoma. It has been suggested that in KS lesions, the lytic cycle gene products may be involved in proliferation of neighboring cells, contributing to pathogenesis. One attractive candidate is an early lytic protein of HHV‐8 that might contribute to oncogenesis is the viral‐encoded G‐protein–coupled receptor of HHV‐8 (ORF74). This protein has been shown to be a viral oncogene and angiogenesis activator. The expression of ORF74 induces an angiogenesis phenotype by secretion of vascular endothelial growth factor, an angiogenesis growth factor. The HHV‐8 ORF74 can also activate mitogen-activated protein kinase, which indicates that the viral protein has a molecular specificity to trigger signaling cascades that are activated by inflammatory cytokines. Another viral protein that may be a candidate for contributing to pathogenesis is the HHV‐8 homologue of human IL‐6, the vIL‐6 protein. Thus, prevention of HHV‐8 replication might provide therapeutic benefits in human disease.
This study by Casper et al. provides evidence that valganciclovir is the first antiviral agent that has been shown to reduce HHV‐8 replication in a randomized clinical trial. The study design was a double blind, placebo‐controlled crossover trial in which 26 HHV‐8 infected men were randomized to receive 8 weeks of valganciclovir or placebo (900 mg once per day administered orally). After a 2‐week washout period, participants received the other study drug for 8 additional weeks. Oral swab samples were taken daily and analyzed for HHV‐8 DNA and CMV DNA by real time PCR. Sixteen HIV‐positive men and 10 HIV‐negative men completed the study. Valganciclovir administered orally effectively inhibited mucosal HHV‐8 replication, as detected by a sensitive PCR assay for HHV‐8 DNA. The antiviral effect of valganciclovir reduced the frequency and quantity of HHV‐8 that was detected in the oropharynx; this effect was prompt and occurred independently of the reduction in CMV replication. The Casper et al. study also provided evidence that HHV‐8 replication occurs independently of CMV replication in immunocompromised patients. The hematologic, renal, and hepatic toxicities of valganciclovir were similar to those of placebo in this short trial of low‐dose valganciclovir.
The Casper et al. study provides important new quantitative data that valganciclovir suppresses replication and oropharyngeal shedding of HHV‐8 and sets the stage for additional research to determine whether valganciclovir prevents Kaposi sarcoma in patients at high risk due to immunosuppression. The effects of valganciclovir on other HHV‐8 associated malignancies, such as primary effusion lymphoma and multicentric Castleman disease, should also be carefully evaluated.
Valganciclovir and Human Herpesvirus–8
Ganciclovir is a nucleoside analogue of guanosine and is the mainstay of treatment against human cytomegalovirus (CMV), a γ‐herpesvirus. Ganciclovir is phosphorylated by herpes simplex virus thymidine kinase or the protein phosphokinase (UL97) of cytomegalovirus to form ganciclovir monophosphate. Cellular kinases then form ganciclovir triphosphate, which is a potent inhibitor of viral DNA polymerase. Ganciclovir triphosphate is an effective inhibitor of CMV replication, but it is not a chain terminator like acyclovir. In the presence of ganciclovir triphosphate, viral DNA replication is greatly slowed, but small fragments of CMV DNA around the origin of DNA replication continue to be made and ganciclovir monophosphate is incorporated into these small fragments of CMV DNA.
In clinical use, human CMV can become resistant to ganciclovir and resistance mutations are found in 2 CMV genes, the UL97 protein phosphokinase and the CMV DNA polymerase. The ganciclovir resistance mutations in the UL97 gene cluster at amino acids 460 and 520, as well as from 590–60. A ganciclovir‐resistant mutation results in a UL97 protein that is unable to phosphorylate ganciclovir. Only 6% of orally administered ganciclovir is bioavailable, which greatly restricts its oral use. By attaching a valine ester to ganciclovir (valganciclovir), the bioavailability of the orally administered drug is greatly increased to 68%. A valine esterase in the human gastrointestinal mucosa cleaves the valine and results in ganciclovir in the portal blood circulation. A daily oral dose of 900 mg of valganciclovir twice per day is equivalent to an intravenously administered dose of ganciclovir of 5 mg/kg twice per day.
The article by Casper et al. in this issue of the Journal provides evidence that valganciclovir can also inhibit replication of human herpesvirus–8 (HHV‐8), another γ herpesvirus. Ganciclovir has been shown to be phosphorylated in the presence of both the HHV‐8 thymidine kinase (open reading frame [ORF] 21) and the HHV‐8 phosphotransferase (ORF36). Two other reports, however, provide conflicting evidence, which suggests that the thymidine kinase of HHV‐8 does not phosphorylate ganciclovir. It is not completely clear, therefore, how ganciclovir is activated to ganciclovir triphosphate during HHV‐8 infection. Phosphorylation by a cellular enzyme remains a possibility. It has also been shown that ganciclovir, cidofovir, and foscarnet inhibit the production of HHV‐8 from latently infected cell lines upon stimulation, whereas acyclovir has little or no activity.
Double‐Edged Genetic Swords and Immunity: Lesson from CCR5 and Beyond. Part 3
Second, the possible consequences of infection with WNV or other flaviviruses in HIV‐positive patients who are receiving CCR5 blockers remains unknown, because very little is understood regarding the long‐term effects of CCR5 blockers on immune functions in vivo. A previous study found that Maraviroc, a CCR5 antagonist, did not influence IL‐2 and CD25 levels, whereas germ‐line inactivation of CCR5 and Ab‐mediated blockade of CCR5 did influence IL‐2 and CD25 levels. This may have been due to differences in the receptor configuration and resulting functionality of Ab‐bound and inhibitor‐bound forms of CCR5. Hence, it is conceivable that the effects on immune function secondary to germ‐line absence of CCR5 in humans and mice versus chemical antagonism of CCR5, such as after administration of Maraviroc, are dissimilar. Given that distinct biological responses of CCR5 might be determined through different receptor conformations, presumably the signaling pathways triggered in cells exposed to Maraviroc versus cells genetically lacking CCR5 may be distinct. Highlighting this possibility is the recent observation that CCR5 forms hetero‐oligomeric complexes with at least 2 other chemokine receptors (CCR2 and CXCR4), and specific antagonists of 1 set of receptors (eg, CCR2 and CCR5) lead to functional cross‐inhibition of the other (ie, CXCR4). This has relevance to the full evaluation of the health consequences of CCR5 blockers, because these data suggest that antagonists of 1 chemokine receptor may regulate the functional properties of another to which they do not directly bind [30]. Thus, Act III may reveal that the immune consequences of CCR5 blockers may not be identical to those found in CCR5‐null people.
Third, the studies by the Murphy group pose a dilemma. Is there a threshold of CCR5 expression, albeit low, that promotes WNV disease? At least in the context of HIV infection, there appears to be a threshold of CCR5 surface expression that is permissive for cell entry, such that small changes in CCR5 density are associated with large increases in HIV infectivity and efficacy of CCR5 blockers. The converse may be operative in WNV infection, in that CCR5 expression levels below a certain (low) threshold may enhance the risk of a more aggressive WNV clinical presentation. Whether such a threshold of CCR5 expression exists is a testable hypothesis because subjects bearing one or lacking the CCR5Δ32 allele display a wide range of CCR5 surface expression levels, and this variability may be partly due to CCR5 promoter polymorphisms that influence expression. Additionally, one may also need to consider other factors that result in low CCR5 expression levels. For example, the copy number of CCL3L1, a potent CCR5 agonist, correlates inversely with CCR5 expression. Hence, Act III may clarify this dilemma.
Finally, we anticipate that Act III will continue to be punctuated by additional examples that show the tradeoffs associated with the CCR5 null state. This is already happening: a recent study showed that CCR5Δ32 homozygosity is associated with increased susceptibility to tick‐borne encephalitis virus. These tradeoffs are a reminder of the constant tug of war between host and pathogen and also of the need to be vigilant, because what we find in one context might differ in another.
Double‐Edged Genetic Swords and Immunity: Lesson from CCR5 and Beyond. Part 2
Murphy’s group showed that after challenge with WNV, CCR5‐null mice had markedly increased viral titers in the central nervous system and had increased mortality compared with that of wild‐type mice, thus suggesting that CCR5 expression was necessary to mount a strong host defense against WNV. Subsequently, they demonstrated that there was a strong epidemiologic association between homozygosity for CCR532 and WNV in humans.
However, because they were unable to distinguish in their previous studies whether the observations were associated with susceptibility to acquiring WNV or associated with the severity of clinical presentation, they conducted the present study. Lim et al now show that the prevalence of CCR5Δ32/Δ32 was comparable in case patients with WNV infection and control participants, which suggests that this genotype is not a susceptibility factor for acquiring WNV infection. However, among the case patients, those patients who were homozygous for CCR532 experienced significantly more symptoms, on average, than did those patients who were heterozygous for CCR532 or who had wild‐type CCR5 genotype. These data indicate that the CCR5 null state is a risk factor for more pronounced early clinical manifestations after infection with WNV.
A noteworthy aspect of the present report by Lim et al is the study design. The case patients and control participants were derived from 35 million blood donors who were screened for WNV. This contrasts with their prior studies, which examined subjects who sought medical attention for symptomatic disease and were compared with otherwise healthy subjects. Also minimizing selection bias, both case patients and control participants in this report were administered the same standardized symptom questionnaire before disclosure of their true WNV infection status, and this study feature facilitated evaluation of the association of the CCR5 null state with the number and severity of early symptoms of WNV infection.
What will Act III reveal? Readers are referred to some possibilities posited in recent opinion pieces. We focus on 4 points. First, the present study raises a pathogenic conundrum: why does the CCR5 null state confer risk for a more aggressive disease but not associate with risk of acquiring WNV infection (ie, viral entry)? One possibility is that CCR5‐mediated signaling events generate critical immune responses that contain the spread of infection but are irrelevant for the initial entry of WNV. In this regard, there are abundant in vitro data linking CCR5 and its ligands to T cell immunity, and 2 recent studies provide corroborative in vivo data: first, that both humans and mice lacking CCR5 surface expression display reduced delayed‐type hypersensitivity skin test responses (an in vivo correlate of T cell function and interleukin 2 [IL‐2] production), and second, that CCR5 expression regulates T cell proliferation, as well as IL‐2 and CD25 expression during T lymphocyte activation. Notably, T cells from CCR5‐null mice secrete lower amounts of IL‐2 than do wild‐type mice; a similar phenotype is observed in CCR5Δ32 homozygotes, as well as after Ab‐mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. These studies underscore that CCR5 expression may influence clinical outcomes after viral infection by affecting parameters (eg, T cell immunity) that are independent of viral entry. This may have relevance to antiviral immune responses to flaviviruses, including WNV, because CD4+ T cells have a critical function in the control and resolution of primary WNV infection; a strong Th1 T cell response, as characterized by interferon and IL‐2 production, results in reduction of neurological sequelae. Thus, one possibility is that the phenotype of “low CCR5 expression–low IL‐2 levels” may contribute to WNV pathogenesis. Hence, we anticipate that Act III will define the precise mechanisms by which CCR5 influences antiviral responses to flaviviruses as well as to lentiviruses.