To Serotype or Not To Serotype: That Is Still the Question
In this issue of the Journal, the report by Banerjee et al. on the postneonatal protective effect of a prior neonatal infection with G10P rotavirus has important implications for rotavirus vaccinology. The protective efficacy of a neonatal rotavirus infection has been addressed previously by various investigators. In a 1983 Australian study, neonates with asymptomatic rotavirus shedding during the first 14 days of life developed fewer and lesssevere rotavirus-positive diarrheal illnesses postneonatally than did control children. The available strains causing neonatal infection had identical electrophoretic patterns (in addition, 1 strain was identified as an “M-like” [G3] strain), whereas the strains causing postneonatal infection had varying patterns. Although serotypic characterization was not readily available at the time, evidence now indicates that asymptomatic neonatal G3 infections protected against community rotavirus strains, including heterotypic serotype G1 and G2 strains, during the 3-year period of surveillance.
Two groups set out to confirmthis concept by comparing the incidence of postneonatal rotavirus illnesses in a cohort of infants infected during the neonatal period with a G9 rotavirus strain 116E or with a G10P strain I321, the latter strain being similar to the neonatal strain described by Banerjee et al. in this issue of the Journal. These studies also found that a neonatal infection induced protection against postneonatal rotavirus illnesses. Data on protection against specific serotypes were not available; however, in the study on strain I321, the postneonatal strains had varying electrophoretic patterns, suggesting heterotypic protection.
A variety of other longitudinal studies of children, beginning at birth and extending up to 3 years of age, have examined the protective efficacy of a primary rotavirus infection against subsequent rotavirus exposure. However, the degree of protection was found to be inconsistent, ranging from none to substantial. Substantial protection was exemplified by a Mexican study in which a single rotavirus infection induced 87% protection against moderate-to-severe rotavirus-positive diarrhea, and 2 infections induced 100% protection. When a second infection occurred, the serotype responsible tended to be different from the original infecting strain. Moreover, high titers of serum anti-rotavirus antibodies, particularly IgA, were associated with protection against rotavirus infection and moderateto- severe diarrhea.
The protective role of preexisting, naturally acquired homotypic and heterotypic rotavirus antibodies was addressed in a study in a home for infants in Japan. A relatively high level of neutralizing antibodies against the infecting serotype was highly protective, whereas neutralizing antibodies against heterotypic strains provided much less or no protection, indicating that homotypic antibodies were a major correlate of protection in this study
The protective effect of neonatal infection on postneonatal illness also has been addressed in vaccine studies in Finland, where neonatal vaccination with a monovalent bovine Nebraska calf diarrhea virus vaccine (NCDV; G6P6) modified the severity of rotavirus illness and a single dose of tetravalent rhesus rotavirus- based tetravalent (RRV-TV) vaccine (a Rotashield precursor) given to neonates protected against fever associated with a second dose of RRV-TV vaccine at 2 months of age.
Analysis of risk factors for mother-to-infant transmission of HCV. Discussion – Part 2
Evidence for the significance of labor-associated factors for the transmission of blood-borne pathogens was already found in investigations of vertical HIV transmission in twin births. First-born twins were more likely to be infected with HIV-1 than their second-born siblings, especially when the former was of greater birth weight. It was suggested in these studies that the first-born twin would have dilated and, to some extent, mechanically cleansed the birth canal, thereby reducing the duration of exposure of the second twin and, consequently, the risk of transmission
Nevertheless, these observations on the transmission of HCV or HIV represent only indirect evidence for an increased risk in vaginal deliveries with prolonged passage through the birth canal. Data on the exact time required for passage of the birth canal, however, are not routinely recorded during delivery and will have to be collected in future prospective clinical trials
Results of studies on HIV infection as a risk factor for vertical HCV transmission also have been ambiguous. Nevertheless, HIV-HCV coinfection reportedly accelerates HCV disease progression by increasing HCV load, and a higher HCV load in HCV-HIV–coinfected patients may be explained by the observed inverse relation between baseline CD4+ cell count and HCV load. In addition, the risk for transmission is exceedingly low in women who are HIV-HCV coinfected but who have little or no detectable HCV RNA. Consequently, we propose that the more important predictive factor for HCV transmission is very likely HCV load, and not HIV coinfection. In the present study, which included only a small number of HIV-HCV–coinfected mothers, no increased risk for vertical HCV transmission was observed in mothers with HIV coinfection. This finding may be explained by the fact that HCV load in our group of HIV-HCV–coinfected mothers did not significantly differ from that in HIV-uninfected mothers.
Reported average mother-to-infant transmission rates of HCV are 5%–10%; we observed a somewhat higher transmission rate of 12%. However, because of the retrospective design of the present study and the fact that HCV infection status could not be assessed for all children, a selection bias cannot be excluded for this parameter. In addition, our laboratory is the major reference laboratory for the geographic region covered by this study; therefore, preselection of patients possibly occurred
In conclusion, vaginal delivery itself does not appear to be a significant risk factor for mother-to-infant transmission of HCV, but the risk of transmission increased with increasing maternal HCV load and with the occurrence of infantile hypoxia or vaginal or perineal lacerations during vaginal delivery. Although it would be premature to recommend routine cesarean section for HCV-infected women, elective cesarean section may reduce the risk of vertical transmission of HCV among mothers with high HCV viremia, those who are at risk for birth injuries during vaginal delivery, or those whose children are at risk for intrapartum hypoxia
Analysis of risk factors for mother-to-infant transmission of HCV. Discussion
Vertical transmission from infected mothers has become the most important mode of HCV infection among children. Although numerous studies have addressed this issue, the mechanisms of vertical HCV transmission, including the timing of infection, remain largely unknown.
It is generally agreed that the risk of vertical HCV infection in mothers without detectable HCV viremia is exceedingly low. We also found HCV viremia to be a prerequisite for transmission, because none of the HCV RNA–negative mothers transmitted the virus to her child.
However, there is no general consensus that the risk of transmission is higher in mothers with high HCV load than in mothers with low HCV load. In the present study, the mean maternal HCV load was higher in mothers whose children were HCV infected than in those whose children were not infected, but high viremia was not a statistically significant risk factor for transmission. However, when we included the mode of delivery into our analysis, it became apparent that, in the case of a vaginal delivery, this difference in mean virus load was even more pronounced. In addition, except for 1 HIV-HCV–coinfected woman, none of the 23 mothers who had a cesarean section transmitted HCV to her offspring
Previous reports found strong evidence that mother-to-child transmission rates and substantial intrapartum transmission of HCV may possibly be reduced by elective cesarean delivery. However, some studies were criticized for testing only a minority of women for HCV RNA and for not addressing possible mechanisms involved in the reduction of vertical transmission of HCV by elective cesarean section. We not only tested 97% of the mothers for HCV RNA but also identified factors that may increase the risk of HCV transmission in vaginal deliveries.
We observed a significantly increased risk of HCV infection in children with a low umbilical cord–blood pH, which is indicative of infantile hypoxia. Whether this intrapartum hypoxia may have led to aspiration of HCV-contaminated maternal fluids or to other hypoxia-related mechanisms of virus transmission can only be speculated.
Moreover, a significantly increased risk of mother-to-infant transmission of HCV was observed in the case of leakage of maternal blood into the birth canal by tears of the cervix or vagina. In contrast, when an episiotomy, which also is associated with maternal bleeding, was performed, it did not increase the risk for vertical transmission of HCV. This apparent contradiction may suggest a more extensive, and possibly longer, exposure of children to virus-contaminated maternal fluids during vaginal deliveries that involve vaginal or perineal lacerations than during those that involve an episiotomy. Although it may be theorized that passage through the birth canal may be prolonged in mothers with relatively large children or that, in women with vaginal or cervical tears, the canal first had to be dilated by the child before passing through it, which allowed for more extensive exposure to virus-contaminated maternal blood, these details will have to be elucidated in prospective clinical trials. Still, since an episiotomy is, in general, performed at the end of labor, intrapartum exposure to maternal blood is very likely shorter in this incidence than in the case of vaginal or perineal lacerations.
Analysis of risk factors for mother-to-infant transmission of HCV
In the present study, vaginal delivery itself did not increase the risk for transmission, compared with cesarean section. However, children who were delivered vaginally and whose mothers sustained a perineal or vaginal laceration had a 6-fold higher risk of becoming HCV-infected than did vaginally-delivered children whose mothers had no laceration. In contrast, no significantly increased risk was observed when an episiotomy was performed. The transmission rate of HCV did not differ between emergency and elective cesarean sections, although the number observed was small
In addition, every reduction in umbilical cord–blood pH by 0.1 increased the risk of mother-to-infant transmission of HCV by 4-fold (assuming a pH standard value of 7.27). An APGAR score of 9/10 at 1 and 5 min was noted for all these HCV-infected newborns
Birth weight of the newborn and gestational age did not increase the risk of mother-to-infant transmission, but we observed a trend for a higher birth weight of the child in women who sustained a perineal or vaginal laceration (mean weight, 2.93 vs. 3.18 kg; estimated relative risk [RR], 3.0; P=.082, logistic regression analysis). No increased risk of mother-to-infant transmission of HCV was observed in primipara or in mothers who experienced a premature rupture of membranes. Six (10%) of 58 mothers did breast-feed, but none of their children was HCV infected
Interactive effects of the risk factors investigated were subsequently evaluated by a multivariate analysis. Results of this analysis were similar to those obtained by univariate logistic regression, except for perineal or vaginal laceration (RR, 2.99; 95% confidence interval, 0.32–27.70) and umbilical cord–blood pH (RR, 5.0; 95% confidence interval, 1.06–23.80)
Follow-up investigation of childrenTo evaluate the course of the HCV infection in infected children and the clearance of maternal HCV antibodies in uninfected children, the 75 children were monitored for as long as possible. All 9 HCV-infected infants were singleton births and were surveyed for a minimum follow-up period of 11 week. The earliest time that 1 newborn was tested and HCV RNA could be detected was at the age of 9 days. Two other children were HCV RNA positive already at the age of 15 and 22 days, respectively. Eight of the 9 HCV-infected children had consistently positive RT-PCR results in the samples collected after the age of 1 month (in 2 children, HCV RNA could be still detected at the age of 5 years). One infected child with detectable HCV RNA became HCV RNA negative at the age of 15 months and remained negative thereafter
All 66 HCV-uninfected children were at least 5 weeks old when last tested for HCV viremia by PCR (median age, 28 weeks; interquartile range, 10–35 weeks). Altogether, 114 samples were collected from these HCV-uninfected children and tested for HCV-specific antibodies by ELISA. When ELISA results were analyzed in relation to the age of the children, a 50% probability of an HCV antibody–negative result was attained at the age of 9.6 months
Mother-to-Infant Transmission of Hepatitis C Virus. Results
Analysis of virological parametersFor the evaluation of possible risk factors for the vertical transmission of HCV, 73 pregnant, HCV-infected women were identified between 1994 and 1999. Serum samples of 71 women (97%) were tested by RT-PCR; 58 women (82%) were HCV RNA positive. Four mothers had been HCV RNA positive before pregnancy but were negative during pregnancy. For 11 mothers, HCV infection was confirmed by the identification of HCV-specific antibodies by use of an immunoblot.
The 73 women gave birth to 75 children, including 2 sets of twins; 9 of the children were HCV infected. Seven (10%) mothers were HIV-HCV–coinfected, 59 (81%) were HIV seronegative, and, for 7 (10%), HIV status could not be determined. For 6 of the 7 HIV-HCV–coinfected mothers, HCV- and HIV-PCRs were done, and both HCV RNA and HIV DNA were detected. Of the 7 mothers with HIV coinfection, 1 transmitted HCV to her offspring and 1 transmitted HIV, but none transmitted both viruses to her child. One of these 7 children (the HIV-infected one) was delivered vaginally, 5 (including the HCV-infected child) were delivered by elective cesarean section, and the mode of delivery was unknown for 1 child.
Analysis of risk factors for mother-to-infant transmission of HCVEstimation of the likelihood of mother-to-infant transmission of HCV for individual virological and clinical parameters was done by unconditional logistic regression analysis. The sample size varied, depending on the number of women for whom data were available for a particular variable Detectable HCV viremia in the mother was a prerequisite for transmission, since only children from mothers with detectable viral RNA became HCV infected. The mothers of infected children had a higher mean HCV load than did mothers of uninfected children (7.3×105 vs. 2.5×104 copies/mL), and this difference in HCV load was even more pronounced among mothers who underwent vaginal delivery (8.1×105 vs. 1.4×104 copies/mL). However, no statistically significant association could be found between maternal HCV RNA level and the risk of mother-to-infant transmission of HCV, although a trend toward a higher risk of transmission with increasing levels of maternal viremia was noted.
In mothers with HIV-HCV coinfection, no increased risk for mother-to-infant transmission of HCV was observed in the present study, although the number investigated was small. In the 7 HIV-HCV–coinfected mothers for whom a quantitative HCV-PCR result was available, the mean HCV load was not significantly higher than that in mothers without HIV-HCV coinfection (1.7×105 vs. 3.2×104 HCV RNA copies/mL; n = 53; P=.113, Mann-Whitney U test).
Mother-to-Infant Transmission of Hepatitis C Virus. Part 3
Testing for HIV-1 antibodies was done with a commercially available HIV-specific ELISA (Abbot HIV 1/2 gO; Abott Diagnostika). If the antibody-ELISA result was positive, it was confirmed by a Western blot (New LAV-BLOT I; Sanofi Diagnostics Pasteur). HIV-1–specific PCRs were done using a commercially available assay (Amplicor HIV-1 Monitor Test; Roche Diagnostic Systems), as recommended by the manufacturer
Clinical investigation Clinical data on HCV-infected mothers were assessed by questionnaires sent to mothers and their gynecologists immediately after delivery and by review of case histories and obstetric notes. The questionnaires asked for the number of previous deliveries, maternal age, and illicit drug use. The case histories and obstetric notes were reviewed for parity of the women, illicit drug use during pregnancy, gestational age, premature rupture of membranes, mode and course of delivery (elective vs. emergency cesarean section and perineal or vaginal laceration), medical interventions during delivery (e.g., episiotomy), umbilical cord–blood pH, APGAR (activity, pulse, grimace, appearance, and respiration) score, and birth weight of the child. Rupture of membranes was considered to be premature if it occurred before the first regular uterine contraction
Drug use was assessed by self-reports and toxicologic testing of urine samples, as described elsewhere. A woman was classified as positive for drug use during pregnancy if she had a positive urine toxicology result or a positive self-report. She was classified as negative for drug use if she had either a negative self-report and a negative urine screen or a negative self-report, if urine toxicology results were unavailable. HCV was reported to have been acquired through IDU for 41 (56%) women and from infected blood or blood products for 6 (8%) women. Other or unknown routes of acquisition were reported for 26 (36%) women. For 4 women with a history of IDU, urine toxicology results were negative during pregnancy, which is indicative of discontinuation of IDU
Data analysisThe effects of the different risk factors for perinatal HCV transmission were evaluated by unconditional logistic regression analysis. For the analysis of HCV load as a risk factor for vertical transmission, HCV-infected women with HCV RNA levels below the limits of detection were assigned a level of 10 copies/mL for logistic regression analysis. Because HCV load may change over time, only quantitative and qualitative HCV-PCR results obtained in samples that were taken within 150 days before and after delivery were used for the estimation of the risk of mother-to-infant transmission of HCV. For the evaluation of deviations from standard umbilical cord–blood pH values, a standard pH value of 7.27 was assumed. Univariate logistic regression results were subsequently checked for robustness by including all pairs of predictors of perinatal HCV transmission, with P<.1, in a multivariate analysis. Because of divergence of estimates, it was not possible to include >2 variables simultaneously in the analysis. P<.05 was considered to be statistically significant. All statistical analyses were performed using SPSS (version 10.0; SPSS) and EGRET (version 2.0.3; Cytel) software
Mother-to-Infant Transmission of Hepatitis C Virus. Part 2
The aim of the present study was to identify preventable risk factors associated with HCV transmission and to provide a basis for the prevention of HCV infection in newborns. For this purpose, we retrospectively evaluated virological and clinical parameters (e.g., mode and course of delivery) within a large, well-defined cohort of HCV-infected pregnant women. Although vaginal delivery itself was not a risk factor for transmission in the present study, we found that perinatal infantile hypoxia and vaginal or perineal laceration that occurred during vaginal delivery significantly increased the risk for HCV transmission.
Patients and Methods
Study populationThe study population included HCV-infected women who were being studied at the Institute of Virology (University of Vienna, Vienna) and who gave birth between 1994 and 1999. HCV infection was identified in these women, before or during pregnancy, by serologic detection of HCV-specific antibodies by use of an ELISA and was confirmed by detection of HCV RNA in serum by use of a reverse-transcriptase polymerase chain reaction (RT-PCR). For the RT-PCR–positive women, HCV load was determined with a quantitative PCR. For the RT-PCR–negative women, the specificity of the antibodies detected by ELISA was confirmed by use of an immunoblot. Furthermore, available serum samples were tested for maternal HIV coinfection according to World Health Organization guidelines. For women without information on HCV load or HIV infection, data were completed by retrospective testing of available serum samples (clinical specimens were stored at −80°C)
The children of these mothers were considered to be HCV uninfected if an HCV-specific RT-PCR done ⩾1 month after birth or an HCV-specific antibody test done ⩾12 months after birth or both were negative. They were considered to be HCV infected if HCV RNA was detected in at least one of their blood samples (umbilical cord blood was not tested)
Children were considered to be HIV-1 uninfected if they were antibody negative on ⩾1 occasion or had at least 2 PCR-negative samples, with at least 1 test done after age 6 months. They were considered to be HIV-1 infected if HIV-1 RNA was detected in at least one of their blood samples. Children with inadequate HIV-1 laboratory data were classified as indeterminate Virological investigationAntibodies to HCV were determined in plasma by ELISA (Monolisa anti-HCV PLUS; Sanofi Diagnostics Pasteur), as recommended by the manufacturer. Before testing of serum samples by PCR, HCV-RNA was extracted with a QIAamp Viral RNA kit (Qiagen). HCV RNA positivity was determined by a qualitative RT-PCR (Amplicor HCV Detection kit [detection limit, 20–1000 copies/mL]; Roche Diagnostic Systems), and HCV load was measured using a quantitative RT-PCR (Amplicor Monitor HCV Assay; Roche Diagnostic Systems). All HCV-PCR–positive results were confirmed by testing a different aliquot of the original sample, and the sensitivity of each qualitative RT-PCR was demonstrated in routine testing by coextraction and codetection of a positive control with ∼1000 copies/mL. For PCR-negative samples, the specificity of ELISA-positive results was confirmed by a recombinant immunoblot (Riba HCV 3.0; Chiron)
Mother-to-Infant Transmission of Hepatitis C Virus
Virological and clinical data from 73 hepatitis C virus (HCV)–infected pregnant women who gave birth to 75 children were merged retrospectively, by logistic regression analysis, to investigate risk factors for vertical transmission of HCV. Eighty-two percent of the HCV-infected mothers were HCV-RNA–positive during pregnancy, and 10% were coinfected with human immunodeficiency virus (HIV). Nine children were HCV infected, 1 was HIV infected, but none was HIV-HCV coinfected. Among vaginal deliveries, the mean HCV load of mothers who transmitted HCV to their infants was higher than that of those who did not (8.1×105 vs. 1.4×104 copies/mL; P=.056). A reduction in umbilical cord–blood pH (relative risk, 3.9; P=.04) or the occurrence of perineal or vaginal laceration (relative risk, 6.4; P=.028) during vaginal delivery significantly increased the risk of vertical HCV transmission. In conclusion, high maternal viremia, infantile hypoxia, and intrapartum exposure to virus-contaminated maternal blood increased the risk of HCV transmission during vaginal deliveries. Consequently, cesarean section may reduce the risk of vertical HCV transmission in selected cases
In developed countries, the majority of new hepatitis C virus (HCV) infections are acquired through injection drug use (IDU). Although vertical transmission of HCV from mother to child during delivery is a rare route of infection, with reported average transmission rates of 5%–10%, it is the predominant mode of infection among children
Until now, only a few risk factors influencing vertical HCV transmission during the perinatal period have been identified (i.e., human immunodeficiency virus [HIV] type 1 coinfection and the presence of HCV RNA in maternal blood). However, it is still controversial whether high maternal virus load also poses a higher risk for transmission. Furthermore, the timing of perinatal transmission is uncertain, and understanding of the obstetrical factors that influence vertical transmission of HCV is still limited. Although several studies did not find an increased risk of transmission during vaginal delivery, a recent investigation of a large cohort found evidence that elective cesarean delivery results in a reduction in mother-to-child transmission rates. Because the factors influencing transmission are still unclear, screening of pregnant women for HCV infection is currently not recommended, and preventive measures during delivery are not available. This uncertainty about risk factors that influence vertical transmission of HCV stands in contrast to mother-to-infant transmission of HIV-1, where risk factors for transmission have been clearly identified (i.e., plasma HIV-1 RNA level and vaginal delivery). As a consequence, medical interventions that significantly reduced vertical transmission rates of HIV-1, including antiviral drugs and elective cesarean section, have been introduced
HIV-1–specific CD8+ T cells can mature to memory in patients receiving ART. Part 5
The results support a dynamic model of T cell differentiation and maturation in well controlled persistent infections. The model is an extension of that proposed by Hamann et al. for T cell development in which CD45RA and CD27 expression was used to distinguish terminally differentiated effector from memory T cells. The extended model proposed here uses CD45RA and CD28 staining to further differentiate between acute and memory CD8+ T cells and proposes that, in well-controlled persistent infection, the acute, memory, and terminally differentiated T cell subsets are not static, as suggested by Appay et al. in a recent linear model of differentiation, but, instead, represent a dynamic system, with flux occurring between these maturation subsets in response to changes in PVL. When the antigen burden is low then, cells mature to memory. During recrudesence, some of these memory cells are recruited back into the acute subset. The distribution of EBV- and CMV-specific T cells across multiple subsets is consistent with this model. Because the distribution of T cells across the different stages of maturation changes in response to fluctuations in antigen load, it can be used as a tool to monitor immune control over the virus.
There is currently considerable interest in the potential use of therapeutic vaccines as adjunctive therapy in HIV-1 infection. It is hoped that by boosting HIV-1–specific immune responses in patients whose infection is well controlled by ART that medication might be stopped either intermittently or perhaps even indefinitely. The results of this study suggest that, although patients receiving ART with low PVLs may be clinically similar, these patients are immunologically heterogenous. Because patients with a high proportion of memory CD8+ T cells might be expected to respond more effectively to immunization than patients with predominantly acute/highly activated T cells that are less able to proliferate, it may be necessary to categorize patients according to the distribution of T cells across the various subsets. It is tempting to hypothesize that one reason for the failure of strategic therapy interruption (STI) trials to exact virologic control while not receiving ART is caused by the possibility that HIV-1–specific CD8+ T cells have not matured to a memory phenotype at the time of STI. A better strategy for future STI or therapeutic immunization studies could be to measure the maturation phenotype of HIV-1–specific CD8+ T cells before planned intervention to be certain that they have progressed to memory.
HIV-1–specific CD8+ T cells can mature to memory in patients receiving ART. Part 4
Although the basis for this generalized failure of HIV-1–specific T cells to mature into terminally differentiated effectors is still not clear, a complete blockade in T cell maturation would predict that the HIV-1–specific CD8+ T cell responses could not mature to memory in patients receiving ART and controlling virus well. It is interesting to note that HIV-1–specific T cells can mature to memory given a sufficient level of control over the virus. Of 11 patients with PVLs <50 HIV RNA copies/mL, as measured by the Ultrasensitive assay, 6 maintained responses that were primarily of an acute phenotype, and 5 had responses with a significant proportion of memory cells. It is important to note that the range of CD8+ T cells within the acute and memory phenotype was 21%–77% and 19%–77%, respectively, and that a difference of >3% was significant in our assay (see Subjects, Materials, and Methods). Longitudinal phenotype measures for some of the patients showed that the HIV-1–specific T cell responses shift toward memory as PVLs decrease and revert to the acute expansion phenotype as PVL increase. For the 11 patients taken together, the ability to shift to memory does not seem to relate to starting PVLs or CD4+ T cell counts but rather to the duration of a high degree of control. Indeed, on the basis of the routine Amplicor and Ultrasensitive PVL measurements for 10 of these patients, we were able to demonstrate that the fraction of HIV-1–specific CD8+ T cells in acute expansion correlated inversely with duration of virus control in patients receiving ART.
Therefore, we find that combined CD45RA/CD27 and CD45RA/CD28 staining can be used effectively to distinguish the acute, effector, and memory subsets in both resolved and persistent infections. This is in contrast to the recent conclusion by Appay et al., who suggested that memory and effector subsets are not accurately described by combined CD27/CD28 staining. This conclusion is based on the observation that, at selected timepoints very early in acute HIV-1 infection (before seroconversion) and in acute EBV and HCV infection, the CD8+ T cells can be mixed for CD28 expression. We believe that this reflects an initial priming period, during which the cells lose CD28 expression as they are being activated. This was clearly shown by Trimble et al., whose detailed study of acute versus convalescent EBV infection indicated that CD28 expression was sequentially lost on expanding CD8+ T cells during the first 10 days of infection. There was a parallel increase in both CD38 and HLA-DR expression, so that the peak of CD38 and HLA-DR expression coincides with the nadir of CD28 expression. A detailed longitudinal analysis of CD28 expression on tetramer-positive cells in HIV-1 infection also shows that, within the acute period of infection, the cells do become primarily CD28−, but this happens over a period of 2–4 weeks. Therefore, in the very early stages of acute viral infection, it appears that the loss of CD28 expression will span the period that it takes to fully activate the cells and that, during this period, combined CD27/CD28 staining may not fully depict all of the cells entering the acute phase. However, once the infection is established and the cells are fully primed, the differentiation states, as defined by these markers, do appear to correspond to acute, memory, and effector CD8+ T cell subsets. There is no doubt that in resolved FLU infection, when antigen is clearly absent or low, the tetramer-positive cells are memory. In well controlled HIV-1 infection, when the PVLs are highly suppressed for an extended duration, the cells approach the same maturation state as that for FLU, and this progression toward maturation correlated with PVL. In the longitudinal analyses of patients commencing or discontinuing ART, the cells are seen to shift toward the memory or acute subsets as the PVL decreases or increases, respectively. These data, coupled with the differences in the dominant maturation state for EBV and CMV tetramer-positive cells, support the view that antigen burden drives the maturation of CD8+ T cells toward the acute, memory, or terminally differentiated effector stages and that the markers accurately reflect these maturation states