key: cord-0008908-77k2nz8h
authors: Gonzalez, Ana María; Jaimes, Maria C.; Rojas, Olga L.; Angel, Juana; Greenberg, Harry B.; A. Franco, Manuel
title: II, 11. Human adaptive immunity to rotaviruses: A model of intestinal mucosal adaptive immunity
date: 2004-09-14
journal: Perspect Med Virol
DOI: 10.1016/s0168-7069(03)09018-9
sha: ee45c12d3b7956e8f2b154f183e3df5d7e355409
doc_id: 8908
cord_uid: 77k2nz8h

This chapter discusses the human adaptive immune response to rotaviruses (RVs), placing the immune response to RVs in the context of the immune response to other mucosal viruses. The chapter discusses the studies of both RV-specific T and B cells. As children with T and/or B immunodeficiencies can develop chronic RV infection, prolonged symptoms, and extraintestinal infection, it is clear that both T and B cells are important for immunity to RV. The various reasons proposed to explain the absence of complete immunity to mucosal viruses such as RV, following primary infection, include a short incubation period after viral exposure, difficulty in maintaining a high level of protective antibody at respiratory and gastrointestinal mucosal surfaces, and a short-lived protective humoral mucosal immune response.

Rotaviruses (RV) are the single most important cause of severe diarrhea in young children, and there is an urgent need to develop effective vaccines against them (Franco and Greenberg, 2001) . Because RV replication is highly restricted to enterocytes in vivo, the immune response against them originates in and exhibits its effector function directly at the intestinal mucosa. For this reason, RV infections axe an excellent model to study intestinal mucosal immunity. Here we will review the human adaptative immune response to RV, first placing the immune response to RV in the context of the immune response to other mucosal viruses, and then highlighting studies of both RV-specific T and B cells.

Since children with T and/or B immunodeficiencies can develop chronic RV infection, prolonged symptoms and extraintestinal infection (Saulsbury et aI., 1980; Wood et al., 1988; Gilger et al., 1992) it is clear that both T and B cells are important for immunity to RV. Nonetheless, the T and B cells induced by natural RV infection are not sufficient in many cases to prevent symptomatic or asymptomatic reinfections in children (Velazquez et al., 1996) or adults (Rodriguez et al., 1987; Nakajima et al., 2001) . The severity and number of RV infections diminish with age, and life threatening infections are generally limited to the primary infection, suggesting that adaptive protective immune responses to RV develop gradually. Reinfection with many other viruses that replicate only at mucosal surfaces is also frequent (Murphy, 1999) . This is in contrast to viruses that spread and replicate systemically for which the development of long lasting protective immunity is the rule (Murphy, 1999) . The various reasons proposed to explain absence of complete immunity to mucosal viruses such as RV, following primary infection, include: 1) A short incubation period after viral exposure. Since the ultimate cellular target for replication of viruses like influenza viruses, rhinoviruses or RV is the same cell as 308 used for entry, these viruses do not require a period of recirculation through the organism. For this reason after secondary viral exposure, the immune system does not have time to amplify the protective specifc responses against these viruses. Sustained high levels of antiviral effector mechanisms at the mucosal surface are thus necessary to achieve protection against these viruses (Roitt, 1997) . 2) Difficulty in maintaining a high level of protective antibody at respiratory and gastrointestinal mucosal surfaces. This problem is due to the high volume, rapid transit time and hostile environment (for example proteases that can inactivate the antibodies) of these surfaces (Murphy, 1999) . 3) A short lived protective humoral mucosal immune response. Although the serum antibody response to systemic viruses (e.g. mumps virus, rubella virus, or HBV) lasts for over a decade, the mucosal antibody responses to influenza virus, respiratory syncytial virus, coronavirus and RV last from 3 to 30 months (Doherty and Ahmed, 1997; Murphy, 1999) . For a given virus (RV included) to which both systemic and mucosal antibody responses develop, the former seem to be more long lasting than the latter (Coulson et al., 1990; Nishio et al., 1990) . 4) Viral mutations that permit the virus to escape the effect of protective neutralizing antibodies. Although this factor is certainly not exclusive to mucosal viruses, it probably plays an important role in conjunction with factors 2 and 3 mentioned above. Protection from reinfections with rhinoviruses seems to be highly dependent on the presence of serotype-specific antibodies but the existence of over 100 viral serotypes makes subsequent infections with viruses of different serotypes highly likely (Doherty and Ahmed, 1997) . Susceptibility to severe influenza disease is also serotype dependent: the appearance of a completely new viral serotype (antigenic shift) causes pandemics. More frequently, minor changes that do not change the serotype of the hemagglutinin (antigenic drift) of a circulating virus seem to be responsible for the susceptibility of individuals to reinfections with influenza virus in each winter epidemic (Doherty and Ahmed, 1997) . For this reason the design of annual influenza vaccines takes into considerations drift mutations in circulating viruses for a given period. RV-specific immunity seems to be, to some extent, serotype-specific (Kapikian et al., 2001) , and reinfections in children appear to be preferentially (but not exclusively) due to viruses of a different serotype (Velazquez et al., 1996) . However, serotype-specific immunity for RVs seems to be less important than for some other mucosal viruses such as rhinoviruses or influenza viruses. Unlike the multitude of serotypes found for rhinoviruses, RV of only five major serotype combinations circulate in most parts of the world (Desselberger et al., 2001) suggesting that variation in viral serotype alone can not account for viral reinfections. In addition, as opposed to influenza virus, the appearance of a new RV serotype (e.g. emerging by reassortment with an animal RV) followed by wide spread disease in a population immune to RV of a different serotype seems to be a very rare event if it exists at all. The appearance of limited mutations in the neutralizing antigens that do not change the viral serotype (like influenza antigenic drift) have been proposed as a factor that could be favoring RV reinfection, but this hypothesis remains unproven at present (Jin et al., 1996; Coulson, 1998 ).

Since serotype-specific immunity cannot completely explain protection against RV, other factors probably play a role in protection against this pathogen. For example, studies in animal models have suggested that non-neutralizing antibodies against other viral proteins (VP6 and or NSP4 (Ball et al., 1996; Burns et al., 1996) ) or other mechanisms (like T cells) can also play a role in immunity (Kapikian et al., 2001) .

The above discussion underscores the importance of studying RV-specific memory T and B lymphocytes, and of investigating the ways these memory cells localize to the intestinal mucosal surfaces were they are required to mediate their protective effect. The understanding of RV-specific CD4+ T cell responses is a key issue because most of RV-specific antibody responses in animal models seem to be dependent on CD4 help (Franco and Greenberg, 1997) . Studies of the T cell response to RV have used lymphoproliferation as a readout after in vitro restimulation with viral antigen (Table 1) . It is generally assumed that results obtained using this method reflect the presence of CD4+ antigen specific T cells. These studies have shown that most, but not all, healthy adults and children and children in the convalescent phase of RV-induced diarrhea have RVspecific CD4+ T cells. The percentage of healthy elderly persons and healthy children below the age of six months that have a lymphoproliferative responses to RV antigen is lower than the percentage of healthy adults and children older than six months of age that have a lymphoproliferative responses to RV antigen (Totterdell et al., 1988a; Offit et al,, 1992) . The T cells that proliferate in response to RV most probably recognize cross-reactive epitopes since restimulation in vitro using simian and bovine RVs as antigens seems to be as efficient as human RV (Table 1 ). The fine specificity of this T cell response, either at a protein or epitope level, has not been thoroughly investigated.

In fact, only one study has formally demonstrated that the proliferative responses were due, at least in part, to CD4 cells (Rott et al., 1997) . * This study used purified subsets of CD4+T lymphocytes and is thus the only one for which the lymphoproliferative activity can directly be attributed to CD4 T cells.

As mentioned above, an important issue to address with RV-specific lymphocytes is the mechanism that permits these lymphocytes to localize and accumulate in the intestinal mucosa were they are required to perform their antiviral effect. Mature naive lymphocytes recirculate between various secondary lymphoid organs until they encounter the antigen that stimulates their specific receptor. After antigen-specific activation, the lymphocyte migration pattern changes, redirecting them preferentially to the tissue where the activating antigen was originally encountered (Butcher et al., 1999) . This tissue-specific migration provides lymphocytes with the best chance of re-encountering the antigen that originally stimulated them. The homing of lymphocytes stimulated by antigens first encountered in intestinal Peyer's patches back to the same or other Peyer's patches or the intestinal lamina propria is mediated by interactions between the integrin c~4~7, expressed on B and T lymphocytes and the cell adhesion molecule MadCAM 1, expressed on the vascular endothelium of the postcapillary venules in the intestine (Butcher et al., 1999) . To date, only one study has addressed the issue of the potential capacity of RV-specific CD4+ T cells from humans to localize to the intestine based on the presence or absence of the intestinal homing receptor (the integrin c~4~7+) on these lymphocytes: Rott et al. studied the ability of c~4~37+ and c~4~7-CD4+ T cell subsets of memory phenotype (CD45RA-) (purified by flow cytometry) to proliferate in response to RV. c~4~7+ CD45RA-cells proliferated more than 2.3 fold over c~4~37-CD45RA-CD4+ T cells to RV antigen (Rott et al., 1997) . In contrast, c~4~37-memory cells were the predominant population responsive to mumps antigen after intramuscular vaccination. These findings suggest that RV-specific T cells, but not T cells specifc for a viral antigen administered parenterally, have the capacity to migrate to the intestinal mucosa (Rott et al., 1997) .

The studies that have used lymphoproliferation to study the T cell response to RV have the limitations that they do not directly quantify or establish the phenotype (with the exception of the study by Rott et al., 1997) of the cells responding to the RV antigen. We have recently adapted a flow cytometry based assay (Waldrop et al., 1997) for the study of RV-specific T cells that detects the intracellular accumulation of cytokines after short term in vitro antigen stimulation (Jaimes et al., 2002) . Because this assay allows to analyse individual cells it permits the simultaneous quantification and characterization of the phenotype of the RV-specific T cells. Using this assay we have been able to characterize, for the first time, human RV-specific CD8+ T cells, and directly determined the frequencies of RV-specific CD4+T cells that secrete IFN-T and IL-13 and CD8+ T cells that secrete IFN-T in children and adults with RV-induced diarrhea and in healthy adults. A representative plot of a flow cytometric analysis of an adult in the convalescent phase of RV induced diarrhea is shown in Fig. 1 . Table  2 shows the frequencies of IFN-7 secreting CD4 and CD8+ RV-specific T cells in healthy adults, and adults and children with RV induced diarrhea: while adults with RV induced diarrhea have greater numbers of RV-specific T cells than healthy adults, children with RV induced diarrhea have very low levels of RV-specific T cells. Using this assay we expect to be able to identify, quantitate and evaluate differences between mucosally primed RV T cells and T cells specific for viruses that replicate systemically. Some future studies will investigate: 1) the pattern and quantity of cytokines Table 2 Frequencies of IFN-y secreting RV-specific CD4 and CD8 T cells in healthy adults and children and adults with RV diarrhea Studies of the h u m a n antibody r e s p o n s e to R V h a v e h i g h l i g h t e d the i m p o r t a n c e of s e r u m I g A ( V e l a z q u e z et al., 2000) and particularly the intestinal I g A response in pro-tection against RV (Coulson et al., 1992) . As for T cells, the detailed characterization of the B cells that produced these IgA antibodies may shed more light on the quantitative and qualitative aspect of intestinal mucosal B cells. Published studies that have characterized these B cells have been limited to the study of effector antibody secreting cells (ASC) identified by ELISPOT. Blood circulating ASC detected in children after acute natural infection or vaccination have been shown to secrete predominantly the IgM isotype (Kaila et al., 1992; Isolauri et al., 1995) . Our current model to explain the detection of these RV-specific ASC in human peripheral blood is that these B cells, originally stimulated by RV antigen in Peyer's patches, are detected in blood during their transit from Peyer's patches, through the circulation and back to the intestine. A recent study supported this model by showing in older children without documented recent RV infection that there exists a correlation between the presence of blood circulating RV-specific ASCs and intestinal ASCs (Brown et al., 2000) . Furthermore, we have recently performed studies similar to those of Rott et al. (1997) but using purified o~4137+ and c~4137-subsets of peripheral blood B cells and then testing in an ELISPOT assay. As expected, RV-specific ASC were predominantly present in the 0~4137+ subpopulation (Gonzalez et al., 2002) .

We have also recently developed a flow cytometry assay in the mouse model that identifies antigen activated (IgD-) B cells (CD19+) expressing surface RV-specific immunoglobulin (Kuklin et al., 2001; Youngman et al., 2002) . With this assay we are able to detect both RV-specific ASCs and memory murine B cells. The principal of this assay consists of the specific binding of a fluorescent RV antigen to a B cell that expresses RV-specific Ig. As an antigen we chose recombinant virus-like particles (VLP) made from RV VP6 protein and a fusion protein consisting of green fluorescent protein (GFP) fused to the N terminus of RV VP2 deleted of its first 92 aminoacids (Charpilienne et al., 2001) . In addition to the RV/GPF-VLPs, the B cells are also stained with fluorochrome labeled monoclonal antibodies directed against a B cell marker (CD19), IgD (to distinguish antigen activated from naive B cells), and the intestinal homing receptor o~4[~7 and then analyzed in a flow cytometer. We have recently applied this assay to detect RV-specific B cells in both children and adults with an acute RV infection (Gonzalez et al., 2002) . An example of one of these experiments is shown in Fig. 2 . Like for RV-specific ASCs, these studies have shown that B cells that express RV-specific surface immunoglobulin from both children and adults predominantly express c~4137 on their surface. These results open the possiblity of characterizing in detail the phenotype of mucosal effector and memory B cells and in particular the expression of other molecules in addition to the integrin c~4[~7 that have been implicated in migration of lymphocytes to the intestine (Cook et al., 2000) . and 1203-04-966-98 and by ICGEB grant COL99-01 (al); by a grant from the National Institutes of Health R37AI21632 and by VA grants to H.B.G.. We would like to thank the subjects that participated in our study for their generosity and the personnel of the pediatrics department of the Hospital San Ignacio for their help in identifying children with diarrhea.

Age-dependent diarrhea induced by a rotaviral nonstructural glycoprotein

Circulating rotavirus-specific antibody-secreting cells (ASCs) predict the presence of rotavirus-specific ASCs in the human small intestinal lamina propria

Protective effect of rotavirus VP6-specific IgA monoclonal antibodies that lack neutralizing activity

Lymphocyte trafficking and regional immunity

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue

Longitudinal studies of neutralizing antibody responses to rotavirus in stools and sera of children following severe rotavirus gastroenteritis

Role of coproantibody in clinical protection of children during reinfection with rotavirus

Comparison of rotavirus immunoglobulin A coproconversion with other indices of rotavirus infection in a longitudinal study in childhood

Rotavirus epidemiology and surveillance

Immune response to viral infection

Immunity to rotavirus in T cell deficient mice

Challenges for rotavirus vaccines

Extraintestinal rotavirus infections in children with immunodeficiency

Rotavirus specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor c~4~37

Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by lactobacillus casei gg

Frequencies of virus specific CD4+ and CD8+ T lymphocytes secreting interferon gamma after acute natural rotavirus infection in children and adults

Divergence of VP7 genes of G1 rotaviruses isolated from infants vaccinated with reassortant rhesus rotaviruses

Preponderance of IgM from blood lymphocytes in response to infantile rotavirus gastroenteritis

Rotaviruses. In: Fields Virology

Protective intestinal anti-rotavirus B cell immunity is dependent on c~4~7 integrin expression but does not require IgA antibody production

Mucosal immunity to viruses

Winter seasonality and rotavirus diarrhoea in adults

Fecal IgA antibody responses after oral poliovirus vaccination in infants and elder children

Rotavirus-specific helper T cell responses in newborns, infants, children, and adults

Longitudinal study of rotavirus infection and gastroenteritis in families served by a pediatric medical practice: Clinical and epidemiologic observations

Immunity to infections

Expression of mucosal homing receptor alpha4beta7 by circulating CD4+ cells with memory for intestinal rotavirus

Chronic rotavirus infection in immunodeficiency

Systemic lymphoproliferative responses to rotavirus

Development of a lymphocyte transformation assay for rotavirus in whole blood and breast milk

Serum antibody as a marker of protection against natural rotavirus infection and disease

Rotavirus infection in infants as protection against subsequent infections

Determination of antigen-specific memory/effector cd4+ t cell frequencies by flow cytometry: Evidence for a novel, antigen-specific homeostatic mechanism in HIV-associated immunodeficiency

Chronic enteric virus infection in two T-cell immunodeficient children

Rotavirus induces proliferative response and augments non-specific cytotoxic activity of lymphocytes in humans

Correlation of tissue distribution, developmental phenotype, and intestinal homing receptor expression of antigen-specific B cells during the murine anti-rotavirus immune response

This work was supported by funds from the Pontificia Universidad Javeriana, ECOS/ ICFES/COLCIENCIAS/ICETEX, Fundaci6n para la Promoci6n de la Investigaci6n y la Tecnologfa, Banco de la Reptiblica grant 1103, Colciencias grants 1203-04-151-98