+-------------+--------------------------------------------------------+
| **          | Each year in the US, approximately 300 children die    |
| Abstract:** | from congenital CMV infection, and at least 9000       |
|             | others suffer serious, permanent disabilities. CMV     |
|             | vaccine development was rated a highest priority in    |
|             | the Institute of Medicine (IOM) report on new vaccine  |
|             | development. The development of a successful CMV       |
|             | vaccine will depend on a thorough understanding of the |
|             | relationship between infection and the host immune     |
|             | response. We wish to determine the frequency of viral  |
|             | shedding in CMV-seropositive women of childbearing     |
|             | age, identify factors associated with shedding,        |
|             | examine how shedding patterns change over time, and    |
|             | determine the frequency with which women are infected  |
|             | with multiple CMV strains. These data will allow us to |
|             | prepare for a future CMV vaccine by 1) estimating the  |
|             | proportion and frequency of infections caused by       |
|             | reactivation versus reinfection; and 2) establishing a |
|             | profile for a host immune response that protects       |
|             | against CMV shedding, thus identifying the type of     |
|             | immune response a vaccine must elicit to be effective. |
+-------------+--------------------------------------------------------+
| **Specific  | *Aim 1*. Estimate the proportion and frequency of CMV  |
| aims:**     | infections caused by reactivation versus reinfection.  |
|             |                                                        |
|             | *Aim 2*. Establish a profile for a host immune         |
|             | response that protects against CMV shedding, thus      |
|             | identifying the type of immune response a vaccine must |
|             | elicit to be effective.                                |
+-------------+--------------------------------------------------------+
| **Ba        | Following primary infection, CMV establishes a latent  |
| ckground:** | infection that allows it to persist indefinitely       |
|             | within its host. Following the establishment of        |
|             | latency, CMV is rarely detected in bodily fluids.      |
|             | Periodically, however, latent virus can reactivate     |
|             | through lytic replication and disseminate beyond its   |
|             | latent target cells. In addition, further exposures to |
|             | CMV-infected individuals can lead to reinfection with  |
|             | a different strain of CMV. In either case, virus may   |
|             | be shed in fluids such as blood, urine, saliva, semen, |
|             | or cervicovaginal specimens. Viral shedding caused by  |
|             | either reactivation of latent virus or reinfection     |
|             | with a different strain is known as recurrent          |
|             | infection.                                             |
|             |                                                        |
|             | A woman who undergoes a primary or recurrent CMV       |
|             | infection during pregancy can transmit that infection  |
|             | to her unborn fetus. Congenital CMV infections in      |
|             | newborns are a leading cause of perinatal              |
|             | complications, including hearing loss,                 |
|             | chorioretinitis, and mental retardation. Each year in  |
|             | the US, approximately 300 children die from congenital |
|             | CMV infection, and at least 9000 others suffer         |
|             | serious, permanent disabilities. CMV vaccine           |
|             | development was rated a highest priority in the        |
|             | Institute of Medicine (IOM) report on new vaccine      |
|             | development \[1\]. Several CMV vaccines are currently  |
|             | under development.                                     |
|             |                                                        |
|             | The development of a successful CMV vaccine will       |
|             | depend on a thorough understanding of the relationship |
|             | between infection and the host immune response.        |
|             | Although the presence of maternal antibody prior to    |
|             | conception reduces the risk of CMV transmission to the |
|             | fetus, acquired maternal "immunity" does not prevent   |
|             | all cases of congenital infection \[2\]. In fact,      |
|             | among populations where most mothers are CMV           |
|             | seropositive, recurrent infection is the most common   |
|             | cause of congenital infection. Whether recurrent       |
|             | infection is usually caused by reactivation or         |
|             | reinfection has important implications for vaccine     |
|             | development. If CMV reactivation is the most common    |
|             | cause of congenital infection, then the safety of a    |
|             | live attentuated vaccine must be scrutinized           |
|             | carefully, but a vaccine would likely prevent          |
|             | congenital infections. If reinfection is the most      |
|             | common cause, then a vaccine must be shown to protect  |
|             | against multiple strains, and behavioral interventions |
|             | for prevention of CMV must continue to be stressed.    |
|             |                                                        |
|             | To better understand the relationship between CMV      |
|             | infection and the immune response, we will determine   |
|             | the frequency of viral shedding in women of            |
|             | childbearing age; identify factors associated with     |
|             | shedding, including host factors such as pregnancy,    |
|             | HIV-related immunosuppresssion, cytokines, and         |
|             | CMV-specific IgG titers and IgM; examine how shedding  |
|             | patterns change over time; and, using genotyping       |
|             | techniques, measure how frequently different viral     |
|             | strains are present. These data will allow us to: 1)   |
|             | identify women who need to be targeted for             |
|             | interventions; 2) estimate the proportion and          |
|             | frequency of recurrent infections caused by            |
|             | reactivation versus reinfection; and 3) establish a    |
|             | profile for a host immune response that protects       |
|             | against CMV shedding, thus identifying the type of     |
|             | immune response a vaccine must elicit to be effective. |
+-------------+--------------------------------------------------------+

+-------------+--------------------------------------------------------+
| **Study     | *Subjects*. We will use specimens from women enrolled  |
| des         | in the HIV Epidemiology Research Study (HERS),         |
| ign/Plan:** | administered by the National Center for HIV, STD, and  |
|             | TB Prevention (NCHSTP) \[3\]. Between 1993 and 1995,   |
| **R         | HERS enrolled 871 HIV-positive women and 439           |
| eferences** | demographically matched and behaviorally comparable    |
|             | HIV-negative women in four cities: Detroit, Baltimore, |
|             | New York, and Providence, Rhode Island. Follow-up      |
|             | visits took place at six-month intervals and continued |
|             | through 2000. At each visit the women received a       |
|             | clinical examination and were asked a wide range of    |
|             | questions regarding drug use and sexual behavior. A    |
|             | previous study found that 92.6% of the women were CMV  |
|             | IgG positive \[4\].                                    |
|             |                                                        |
|             | *Laboratory testing*. We will measure CMV IgG antibody |
|             | titers and IgM in serum using well-established         |
|             | techniques. Cytokine testing will also be done on      |
|             | serum to measure levels of various cytokines related   |
|             | to T~H~1, T~H~2, pro-inflammatory, or                  |
|             | anti-inflammatory immune responses. TaqMan PCR will be |
|             | done to quantify CMV DNA load in peripheral blood      |
|             | mononuclear cells (PBMC) and cervicovaginal lavage     |
|             | (CVL) specimens. CMV genotyping will be done on        |
|             | PCR-positive specimens, focusing on recognized         |
|             | variable sequences in the gB and gN genes of CMV.      |
|             |                                                        |
|             | *Testing algorithm.* Testing will proceed in two       |
|             | phases. First, we will take a cross-sectional sample   |
|             | of 500 CMV IgG-positive women to examine frequency of  |
|             | and risk factors for viral shedding in PBMC and CVL.   |
|             | The sample will be stratified by HIV status and CD4    |
|             | count. HIV-negative women will be oversampled since we |
|             | expect fewer of them to be shedding CMV. In the second |
|             | phase, we will carry out longitudinal testing of women |
|             | who were CMV PCR positive in phase one, along with a   |
|             | random sample of PCR-negative women. Cytokines, CMV    |
|             | DNA, IgG titers and IgM will be measured in both       |
|             | phases; CMV genotyping will be done on PCR-positive    |
|             | specimens from phase two. Overall, close to 10,000     |
|             | different tests will be run.                           |
|             |                                                        |
|             | *Analyses.* Statistical methods such as logistic       |
|             | regression will be used to identify risk factors for   |
|             | CMV shedding. Longitudinal analyses will be carried    |
|             | out using generalized estimating equations. Sequence   |
|             | analysis will be performed as follows: the             |
|             | sample-derived sequences will be aligned with the      |
|             | corresponding regions of the reference CMV strains     |
|             | using the Wisconsin GCG Sequence Analysis Package,     |
|             | version 10. Phylogenetic analyses will be performed    |
|             | using the DNADIST (Kimura two-parameter method),       |
|             | Neighbor (neighbor-joining method), SEQBOOT, and       |
|             | consense programs of PHYLIP package, version 3.5. The  |
|             | TREEVIEW software will be used to generate             |
|             | phylogenetic trees.                                    |
|             |                                                        |
|             | ####### 1. Institute of Medicine Report. Vaccines f    |
|             | or the 21st century: a tool for decision making, 1999. |
|             |                                                        |
|             | 2\. Fowler KB et al. *JAMA* 2003, 289:1008-1011.       |
|             |                                                        |
|             | 3\. Smith DK et al. *Am J Epidemiol* 1997,             |
|             | 146:459-469.                                           |
|             |                                                        |
|             | 4\. Stover CT et al. *J Infect Dis* 2003,              |
|             | 187:1388-1396.                                         |
+-------------+--------------------------------------------------------+