Comparison of the Sensitivity and Specificity of Commercial Anti-Dengue Virus IgG Tests to Identify Persons Eligible for Dengue Vaccination.

The Advisory Committee on Immunization Practices (ACIP) recommended that dengue pre-vaccination screening tests for Dengvaxia administration have at least 98% specificity and 75% sensitivity. This study evaluates the performance of commercial anti-DENV IgG tests to identify tests that could be used for pre-vaccination screening. First, for 7 tests, we evaluated sensitivity and specificity in early convalescent dengue virus (DENV) infection, using 44 samples collected 7-30 days after symptom onset and confirmed by RT-PCR. Next, for the 5 best performing tests and two additional tests (with and without an external test reader) that became available later, we evaluated performance to detect past dengue infection among a panel of 44 specimens collected in 2018-2019 from healthy 9-16-year-old children from Puerto Rico. Finally, a full-scale evaluation was done with the 4 best performing tests using 400 specimens from the same population. We used virus focus reduction neutralization test and an in-house DENV IgG ELISA as reference standards. Of seven tests, five showed ≥75% sensitivity detecting anti-DENV IgG in early convalescent specimens with low cross-reactivity to Zika virus. For the detection of previous DENV infections the tests with the highest performance were the Euroimmun NS1 IgG ELISA (sensitivity 84.5%, specificity 97.1%) and CTK Dengue IgG rapid test R0065C with the test reader (sensitivity 76.2% specificity 98.1%). There are IgG tests available that can be used to accurately classify individuals with previous DENV infection as eligible for dengue vaccination to support safe vaccine implementation.

Previous Dengue Infection among Children in Puerto Rico and Implications for Dengue Vaccine Implementation.

Limited dengue virus (DENV) seroprevalence estimates are available for Puerto Rico, which are needed to inform the potential use and cost-effectiveness of DENV vaccines. The Communities Organized to Prevent Arboviruses (COPA) is a cohort study initiated in 2018 in Ponce, Puerto Rico, to assess arboviral disease risk and provide a platform to evaluate interventions. We recruited participants from households in 38 study clusters, who were interviewed and provided a serum specimen. Specimens from 713 children aged 1 to 16 years during the first year of COPA were tested for the four DENV serotypes and ZIKV using a focus reduction neutralization assay. We assessed the seroprevalence of DENV and ZIKV by age and developed a catalytic model from seroprevalence and dengue surveillance data to estimate the force of infection for DENV during 2003-2018. Overall, 37% (n = 267) were seropositive for DENV; seroprevalence was 9% (11/128) among children aged 1 to 8 years and 44% (256/585) among children aged 9 to 16 years, exceeding the threshold over which DENV vaccination is deemed cost-effective. A total of 33% were seropositive for ZIKV, including 15% among children aged 0 to 8 years and 37% among children aged 9 to 16 years. The highest force of infection occurred in 2007, 2010, and 2012-2013, with low levels of transmission from 2016 to 2018. A higher proportion of children had evidence of multitypic DENV infection than expected, suggesting high heterogeneity in DENV risk in this setting.

Epidemiologic Trends of Dengue in U.S. Territories, 2010-2020.

Problem/Condition: Dengue is one of the most common vectorborne flaviviral infections globally, with frequent outbreaks in tropical regions. In 2019 and 2020, the Pan American Health Organization reported approximately 5.5 million dengue cases from the Americas, the highest number on record. In the United States, local dengue virus (DENV) transmission has been reported from all U.S. territories, which are characterized by tropical climates that are highly suitable for Aedes species of mosquitoes, the vector that transmits dengue. Dengue is endemic in the U.S. territories of American Samoa, Puerto Rico, and the U.S. Virgin Islands (USVI). Dengue risk in Guam and the Commonwealth of the Northern Mariana Islands is considered sporadic or uncertain. Despite all U.S. territories reporting local dengue transmission, epidemiologic trends over time have not been well described. Reporting Period: 2010-2020. Description of System: State and territorial health departments report dengue cases to CDC through ArboNET, the national arboviral surveillance system, which was developed in 2000 to monitor West Nile virus infections. Dengue became nationally notifiable in ArboNET in 2010. Dengue cases reported to ArboNET are categorized using the 2015 Council of State and Territorial Epidemiologists case definition. In addition, DENV serotyping is performed at CDC's Dengue Branch Laboratory in a subset of specimens to support identification of circulating DENV serotypes. Results: During 2010-2020, a total of 30,903 dengue cases were reported from four U.S. territories to ArboNET. Puerto Rico reported the highest number of dengue cases (29,862 [96.6%]), followed by American Samoa (660 [2.1%]), USVI (353 [1.1%]), and Guam (28 [0.1%]). However, annual incidence rates were highest in American Samoa with 10.2 cases per 1,000 population in 2017, followed by Puerto Rico with 2.9 in 2010 and USVI with 1.6 in 2013. Approximately one half (50.6%) of cases occurred among persons aged <20 years. The proportion of persons with dengue who were hospitalized was high in three of the four territories: 45.5% in American Samoa, 32.6% in Puerto Rico, and 32.1% in Guam. In Puerto Rico and USVI, approximately 2% of reported cases were categorized as severe dengue. Of all dengue-associated deaths, 68 (0.2%) were reported from Puerto Rico; no deaths were reported from the other territories. During 2010-2020, DENV-1 and DENV-4 were the predominant serotypes in Puerto Rico and USVI. Interpretation: U.S. territories experienced a high prevalence of dengue during 2010-2020, with approximately 30,000 cases reported, and a high incidence during outbreak years. Children and adolescents aged <20 years were disproportionately affected, highlighting the need for interventions tailored for this population. Ongoing education about dengue clinical management for health care providers in U.S. territories is important because of the high hospitalization rates reported. Dengue case surveillance and serotyping can be used to guide future control and prevention measures in these areas. Public Health Action: The Advisory Committee on Immunization Practices recommends vaccination with Dengvaxia for children aged 9-16 years with evidence of previous dengue infection and living in areas where dengue is endemic. The recommendation for the dengue vaccine offers public health professionals and health care providers a new intervention for preventing illness and hospitalization in the age group with the highest burden of disease in the four territories (Paz Bailey G, Adams L, Wong JM, et al. Dengue Vaccine: Recommendations of the Advisory Committee on Immunization Practices, United States, 2021. MMWR Recomm Rep 2021;70[No. RR-6]). American Samoa, Puerto Rico, and USVI are all considered endemic areas and persons residing in these areas are eligible for the new dengue vaccine. Persons aged 9-16 years in those jurisdictions with laboratory evidence of previous dengue infection can receive the dengue vaccine and benefit from a reduced risk for symptomatic disease, hospitalization, or severe dengue. Health care providers in these areas should be familiar with the eligibility criteria and recommendations for vaccination to reduce the burden of dengue among the group at highest risk for symptomatic illness. Educating health care providers about identification and management of dengue cases can improve patient outcomes and improve surveillance and reporting of dengue cases.

Risk factors for infection with chikungunya and Zika viruses in southern Puerto Rico: A community-based cross-sectional seroprevalence survey.

Chikungunya virus (CHIKV) caused a large outbreak in Puerto Rico in 2014, followed by a Zika virus (ZIKV) outbreak in 2016. Communities Organized for the Prevention of Arboviruses (COPA) is a cohort study in southern Puerto Rico, initiated in 2018 to measure arboviral disease risk and provide a platform to evaluate interventions. To identify risk factors for infection, we assessed prevalence of previous CHIKV infection and recent ZIKV and DENV infection in a cross-sectional study among COPA participants. Participants aged 1-50 years (y) were recruited from randomly selected households in study clusters. Each participant completed an interview and provided a blood specimen, which was tested by anti-CHIKV IgG ELISA assay and anti-ZIKV and anti-DENV IgM MAC-ELISA assays. We assessed individual, household, and community factors associated with a positive result for CHIKV or ZIKV after adjusting for confounders. During 2018-2019, 4,090 participants were enrolled; 61% were female and median age was 28y (interquartile range [IQR]: 16-41). Among 4,035 participants tested for CHIKV, 1,268 (31.4%) had evidence of previous infection. CHIKV infection prevalence was lower among children 1-10 years old compared to people 11 and older (adjusted odds ratio [aOR] 2.30; 95% CI 1.71-3.08). Lower CHIKV infection prevalence was associated with home screens (aOR 0.51; 95% CI 0.42-0.61) and air conditioning (aOR 0.64; 95% CI 0.54-0.77). CHIKV infection prevalence also varied by study cluster of residence and insurance type. Few participants (16; 0.4%) had evidence of recent DENV infection by IgM. Among 4,035 participants tested for ZIKV, 651 (16%) had evidence of recent infection. Infection prevalence increased with older age, from 7% among 1-10y olds up to 19% among 41-50y olds (aOR 3.23; 95% CI 2.16-4.84). Males had an increased risk of Zika infection prevalence compared with females (aOR 1.31; 95% CI 1.09-1.57). ZIKV infection prevalence also decreased with the presence of home screens (aOR 0.66; 95% CI 0.54-0.82) and air conditioning (aOR 0.69; 95% CI 0.57-0.84). Similar infection patterns were observed for recent ZIKV infection prevalence and previous CHIKV infection prevalence by age, and the presence of screens and air conditioners in the home decreased infection risk from both viruses by as much as 50%.

Capacity of a Multiplex IgM Antibody Capture ELISA to Differentiate Zika and Dengue Virus Infections in Areas of Concurrent Endemic Transmission.

Serological cross-reactivity has proved to be a challenge to diagnose Zika virus (ZIKV) infections in dengue virus (DENV) endemic countries. Confirmatory testing of ZIKV IgM positive results by plaque reduction neutralization tests (PRNTs) provides clarification in only a minority of cases because most individuals infected with ZIKV were previously exposed to DENV. The goal of this study was to evaluate the performance of a ZIKV/DENV DUO IgM antibody capture ELISA (MAC-ELISA) for discriminating between DENV and ZIKV infections in endemic regions. Our performance evaluation included acute and convalescent specimens from patients with real-time reverse transcription polymerase chain reaction (RT-PCR)-confirmed DENV or ZIKV from the Sentinel Enhanced Dengue Surveillance System in Ponce, Puerto Rico. The ZIKV/DENV DUO MAC-ELISA specificity was 100% for DENV (N = 127) and 98.4% for ZIKV (N = 275) when specimens were tested during the optimal testing window (days post-onset of illness [DPO] 6-120). The ZIKV/DENV DUO MAC-ELISA sensitivity of RT-PCR confirmed specimens reached 100% for DENV by DPO 6 and for ZIKV by DPO 9. Our new ZIKV/DENV DUO MAC-ELISA was also able to distinguish ZIKV and DENV regardless of previous DENV exposure. We conclude this novel serologic diagnostic assay can accurately discriminate ZIKV and DENV infections. This can potentially be useful considering that the more labor-intensive and expensive PRNT assay may not be an option for confirmatory diagnosis in areas that lack PRNT capacity, but experience circulation of both DENV and ZIKV.

Novel Assay to Measure Seroprevalence of Zika Virus in the Philippines.

Zika virus (ZIKV) is a member of the Flaviviridae family, which includes other clinically notable viruses such as the 4 dengue virus serotypes (DENV-1-4). Distinguishing DENVs from ZIKV using the established serologic assays widely used for monitoring DENV transmission is difficult because of antibody cross-reactivity between these closely related flaviviruses. We describe a modified and improved recombinant envelope domain III-based serologic assay for detecting ZIKV type-specific antibodies in regions with endemic DENV transmission. When the assay was used to measure ZIKV seroprevalence in 2017 among children 9-14 years of age living in a region of the Philippines with endemic DENV transmission, we observed a ZIKV seroprevalence of 18%. Investigators should consider using the ZIKV envelope domain III-based assay, which is simple and readily adaptable for use in standard clinical and public health laboratories, to assess ZIKV seroprevalence in areas with endemic DENV transmission.

Evaluating Differences in Whole Blood, Serum, and Urine Screening Tests for Zika Virus, Puerto Rico, USA, 2016.

We evaluated nucleic acid amplification testing (NAAT) for Zika virus on whole-blood specimens compared with NAAT on serum and urine specimens among asymptomatic pregnant women during the 2015-2016 Puerto Rico Zika outbreak. Using NAAT, more infections were detected in serum and urine than in whole blood specimens.

Dengue Virus Infections among Peace Corps Volunteers in Timor-Leste, 2018-2019.

Dengue is an ongoing health risk for Peace Corps Volunteers (PCVs) working in the tropics. On May 2019, the Peace Corps Office of Health Services notified the Centers for Disease Control and Prevention (CDC) of a dengue outbreak among PCVs in Timor-Leste. The purpose of this investigation was to identify the clinical, demographic, and epidemiological characteristics of PCVs with dengue and recommend dengue preventive measures. To identify PCVs with dengue and describe disease severity, the medical records of PCVs reporting fever during September 2018-June 2019 were reviewed. To identify factors associated with dengue virus (DENV) infection, we administered a questionnaire on demographics, travel history, and mosquito avoidance behaviors and collected blood specimens to detect the anti-DENV IgM antibody to diagnose recent infection. Of 35 PCVs in-country, 11 (31%) tested positive for dengue (NS1, IgM, PCR), eight requiring hospitalization and medical evacuation. Among 27 (77%) PCVs who participated in the investigation, all reported having been recently bitten by mosquitoes and 56% reported being bitten most often at home; only 16 (59%) reported having screens on bedroom windows. Nearly all (93%) PCVs reported using a bed net every night; fewer (70%) reported using mosquito repellent at least once a day. No behaviors were significantly associated with DENV infection. Raising awareness of dengue risk among PCVs and continuing to encourage mosquito avoidance behavior to prevent dengue is critical. Access to and use of measures to avoid mosquito bites should be improved or implemented. Peace Corps medical officers should continue to receive an annual refresher training on dengue clinical management.

Laboratory-Acquired Dengue Virus Infection, United States, 2018.

Investigation of a dengue case in a laboratory worker in North Carolina, USA, revealed that the case-patient prepared high-titer dengue virus stocks soon before illness onset. Improper doffing of gloves with an open finger wound likely resulted in cutaneous exposure. This case reinforces recommendations for enhanced precautions when working with high-titer dengue virus.

Entomological Investigation Detects Dengue Virus Type 1 in Aedes (Stegomyia) albopictus (Skuse) during the 2015-16 Outbreak in Hawaii.

A dengue outbreak occurred on Hawaii Island between September 2015 and March 2016. Entomological investigations were undertaken between December 2015 and February 2016 to determine which Aedes mosquito species were responsible for the outbreak. A total of 3,259 mosquitoes were collected using a combination of CDC autocidal gravid ovitraps, Biogents BG-Sentinel traps, and hand-nets; immature mosquitoes were collected during environmental surveys. The composition of species was Aedes albopictus (58%), Aedes aegypti (25%), Wyeomyia mitchelli (7%), Aedes vexans (5%), Culex quinquefasciatus (4%), and Aedes japonicus (1%). Adult mosquitoes were analyzed by real-time reverse transcription polymerase chain reaction (PCR) for the presence of dengue virus (DENV) RNA. Of the 185 pools of female mosquitoes tested, 15 containing Ae. albopictus were positive for the presence of DENV type 1 RNA. No virus was detected in pools of the remaining species. Phylogenetic analysis showed the virus strain belonged to genotype I and was closely related to strains that were circulating in the Pacific between 2008 and 2014. This is the first report of detection of DENV in Ae. albopictus from Hawaii.

Duration of the Presence of Infectious Zika Virus in Semen and Serum.

Zika virus (ZIKV) has recently caused a large epidemic in the Americas that is associated with birth defects. Although ZIKV is primarily transmitted by Aedes mosquitoes, ZIKV RNA is detectable in blood and semen of infected individuals for weeks or months, during which sexual and other modes of transmission are possible. However, viral RNA is usually detectable longer than infectious virus is present. We determined the frequency of isolation of infectious virus from semen and serum samples prospectively obtained from a cohort of patients in Puerto Rico. We confirmed isolation of infectious virus on the basis of a tissue culture cytopathic effect, an increase in virus genome copy equivalents (GCE), and positive results of immunofluorescence analysis; virus in infected cells was quantitated by flow cytometry. These criteria confirmed the presence of infectious virus in semen specimens from 8 of 97 patients for up to 38 days after initial detection when virus loads are >1.4 × 106 genome copy equivalents/mL. Two serum isolates were obtained from 296 patients. These findings can help guide important prevention guidelines for persons that may potentially be infectious and transmit ZIKV sexually.

Comprehensive Evaluation of Differential Serodiagnosis between Zika and Dengue Viral Infections.

Diagnostic testing for Zika virus (ZIKV) or dengue virus (DENV) infection can be accomplished by a nucleic acid detection method; however, a negative result does not exclude infection due to the low virus titer during infection depending on the timing of sample collection. Therefore, a ZIKV- or DENV-specific serological assay is essential for the accurate diagnosis of patients and to mitigate potential severe health outcomes. A retrospective study design with dual approaches of collecting human serum samples for testing was developed. All serum samples were extensively evaluated by using both noninfectious wild-type (wt) virus-like particles (VLPs) and soluble nonstructural protein 1 (NS1) in the standard immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Both ZIKV-derived wt-VLP- and NS1-MAC-ELISAs were found to have similar sensitivities for detecting anti-premembrane/envelope and NS1 antibodies from ZIKV-infected patient sera, although lower cross-reactivity to DENV2/3-NS1 was observed. Furthermore, group cross-reactive (GR)-antibody-ablated homologous fusion peptide-mutated (FP)-VLPs consistently showed higher positive-to-negative values than homologous wt-VLPs. Therefore, we used DENV-2/3 and ZIKV FP-VLPs to develop a novel, serological algorithm for differentiating ZIKV from DENV infection. Overall, the sensitivity and specificity of the FP-VLP-MAC-ELISA and the NS1-MAC-ELISA were each higher than 80%, with no statistical significance. The accuracy can reach up to 95% with the combination of FP-VLP and NS1 assays. In comparison to current guidelines using neutralization tests to measure ZIKV antibody, this approach can facilitate laboratory screening for ZIKV infection, especially in regions where DENV infection is endemic and capacity for neutralization testing does not exist.

Persistence of Zika Virus in Body Fluids - Final Report.

BACKGROUND: To estimate the frequency and duration of detectable Zika virus (ZIKV) RNA in human body fluids, we prospectively assessed a cohort of newly infected participants in Puerto Rico. METHODS: We evaluated samples obtained from 150 participants (including 55 men) in whom ZIKV RNA was detected on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay in urine or blood in an enhanced arboviral clinical surveillance site. We collected serum, urine, saliva, semen, and vaginal secretions weekly for the first month and then at 2, 4, and 6 months. All specimens were tested by means of RT-PCR, and serum was tested with the use of anti-ZIKV IgM enzyme-linked immunosorbent assay. Among the participants with ZIKV RNA in any specimen at week 4, biweekly collection continued until all specimens tested negative. We used parametric Weibull regression models to estimate the time until the loss of ZIKV RNA detection in each body fluid and reported the findings in medians and 95th percentiles. RESULTS: The medians and 95th percentiles for the time until the loss of ZIKV RNA detection were 14 days (95% confidence interval [CI], 11 to 17) and 54 days (95% CI, 43 to 64), respectively, in serum; 8 days (95% CI, 6 to 10) and 39 days (95% CI, 31 to 47) in urine; and 34 days (95% CI, 28 to 41) and 81 days (95% CI, 64 to 98) in semen. Few participants had detectable ZIKV RNA in saliva or vaginal secretions. CONCLUSIONS: The prolonged time until ZIKV RNA clearance in serum in this study may have implications for the diagnosis and prevention of ZIKV infection. Current sexual-prevention guidelines recommend that men use condoms or abstain from sex for 6 months after ZIKV exposure; in 95% of the men in this study, ZIKV RNA was cleared from semen after about 3 months. (Funded by the Centers for Disease Control and Prevention.).

Multiplexed Biomarker Panels Discriminate Zika and Dengue Virus Infection in Humans.

Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses that cause widespread, acute febrile illnesses, notably microcephaly for fetuses of infected pregnant women. Detecting the viral cause of these illnesses is paramount to determine risks to patients, counsel pregnant women, and help fight outbreaks. A combined diagnostic algorithm for ZIKV and DENV requires Reverse transcription polymerase chain reaction (RT-PCR) and IgM antibody detection. RT-PCR differentiates between DENV and ZIKV infections during the acute phases of infection, but differentiation based on IgM antibodies is currently nearly impossible in endemic areas. We have developed a ZIKV/DENV protein array and tested it with serum samples collected from ZIKV- and DENV-infected patients and healthy subjects in Puerto Rico. Our analyses reveal a biomarker panel that are capable of discriminating ZIKV and DENV infections with high accuracy, including Capsid protein from African ZIKV strain MR766, and other 5 pair of proteins (NS1, NS2A, NS3, NS4B and NS5) from ZIKV and DENV respectively. Both sensitivity and specificity of the test for ZIKV from DENV are around 90%. We propose that the ZIKV/DENV protein array will be used in future studies to discriminate patients infected with ZIKV from DENV.

Differences in type I interferon signaling antagonism by dengue viruses in human and non-human primate cell lines.

BACKGROUND/OBJECTIVES: In vitro studies have shown that dengue virus (DENV) can thwart the actions of interferon (IFN)-α/ß and prevent the development of an antiviral state in infected cells. Clinical studies looking at gene expression in patients with severe dengue show a reduced expression of interferon stimulated genes compared to patients with dengue fever. Interestingly, there are conflicting reports as to the ability of DENV or other flaviviruses to inhibit IFN-α/ß signaling. METHODOLOGY/PRINCIPAL FINDINGS: In order to determine the relative inhibition of IFN-α/ß signaling by DENVs, a method combining flow cytometry and a four-parameter logistic regression model was established. A representative isolate from DENV-1, -3 and -4 and seventeen representative isolates encompassing all DENV-2 genotypes were evaluated. All of the DENVs evaluated in this study were capable of inhibiting IFN-α/ß signaling. Most of the strains were able to inhibit IFN-α/ß to a degree similar to DENV strain 16681; however, DENV-2 sylvatic strains demonstrated an increased inhibition of phosphorylated signal transducer and activator of transcription (pSTAT1). Surprisingly, we were unable to observe inhibition of pSTAT1 by DENV-2 sylvatic strains or the Asian strain 16681 in non-human primate (NHP) cell lines. Analysis in primary Rhesus macaque dendritic cells suggests that DENVs are capable of inhibiting IFN signaling in these cells. However, contrary to human dendritic cells, production of IFN-α was detected in the supernatant of DENV-infected Rhesus macaque dendritic cells. CONCLUSIONS: The ability of DENVs to inhibit IFN-α/ß signaling is conserved. Although some variation in the inhibition was observed, the moderate differences may be difficult to correlate with clinical outcomes. DENVs were unable to inhibit pSTAT1 in NHP cell lines, but their ability to inhibit pSTAT1 in primary Rhesus macaque dendritic cells suggests that this may be a cell specific phenomena or due to the transformed nature of the cell lines.

Impairment of CD4+ T cell polarization by dengue virus-infected dendritic cells.

BACKGROUND: The production of type I interferon alpha/beta (IFN-α/ß) is crucial to viral clearance during dengue virus (DENV) infection; however, in vitro-infected dendritic cells (DCs) exhibit a decreased capacity to respond to IFN-α/ß stimulation, and antigen-presenting cells (APCs) isolated from patients with acute DENV infection exhibit defects in T cell priming. METHODS: In order to ascertain the stimulatory capacity of primary human monocyte-derived DCs infected with wild-type DENV isolates, representing a range of genotypes and disease outcomes, we cocultured infected DCs with allogeneic-naive CD4(+) T cells. The gene expression patterns of IFN-α/ß sensitive genes were quantitated to determine if the infected DCs displayed a blunted IFN-α/ß response. RESULTS: DENV-infected DCs induced the initial proliferation of naive CD4(+) T cells but they remained nonpolarized in effector function. The expression of IFN-α/ß-stimulated genes was downregulated, revealing that the inhibition of IFN-α/ß signaling is conserved among endemic DENV serotype 2 strains. CONCLUSIONS: The failure of naive CD4(+) T cells to differentiate into IFN gamma-producing effector T cells when primed by DENV-infected DCs cannot be explained solely by a block in IFN-α/ß signaling, suggesting that the ability of DENV to evade the early host response is multifaceted.

Immune dysfunction in caveolin-1 null mice following infection with Trypanosoma cruzi (Tulahuen strain).

In recent years, host cell caveolae/caveolins have emerged as potentially important targets for pathogenic microorganisms; therefore, we investigated the role of caveolin-1 (Cav-1) in T. cruzi infection using Cav-1 null mice. Cav-1 null and wild type mice were infected with the virulent Tulahuen strain. The mortality was 100% in both groups, but death was slightly delayed in wild type mice. The parasitemia in the Cav-1 null mice was significantly reduced compared with wild type littermates. Histopathologic examination of the heart revealed numerous pseudocysts, myonecrosis, and marked inflammation, which was similar in both mouse groups. Real-time PCR confirmed these observations. Infection of cultured cardiac fibroblasts obtained from Cav-1 null and wild type mice revealed no differences in infectivity. Determination of serum levels of several inflammatory mediators revealed a striking reduction in IFN-gamma, TNF-alpha and components of the nitric oxide pathway in infected Cav-1 null mice. Infection of wild type mice resulted in the expected enhancement of inflammatory mediators. The defective production of chemokines and cytokines observed in vivo is in part attributed to Cav-1 null macrophages. Despite these marked differences in the response to infection by inflammatory mediators between the two mouse strains, the final outcome was similar. These results suggest that Cav-1 may play an important role in the normal development of immune responses.

Caveolin-1-deficient mice show defects in innate immunity and inflammatory immune response during Salmonella enterica serovar Typhimurium infection.

A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in Salmonella pathogenesis. Cav-1(-/-) mice displayed a significant decrease in survival when challenged with Salmonella enterica serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1(-/-) mice. However, infection of Cav-1(-/-) macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1(-/-) mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1(-/-) mice were unable to control the systemic infection of Salmonella. The increased chemokine production in Cav-1(-/-) mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1(-/-) macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to Salmonella LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to S. enterica serovar Typhimurium.

A novel role for caveolin-1 in B lymphocyte function and the development of thymus-independent immune responses.

Caveolin-1 (Cav-1) functions as a scaffold or platform for many molecules involved in signal transduction. However, the expression and function of Cav-1 in the immune system has been controversial. Here, we show that Cav-1 mRNA and protein is indeed expressed in murine B-lymphocytes in a regulated mannerin response to LPS. Cav-1 deficient mice displayed reduced levels of antibody in their serum. In order to examine the role of Cav-1 in the development of immunoglobulin-mediated immune responses, we immunized wild-type and Cav-1 deficient mice with thymus-dependent and thymus independent antigens. Our results show that Cav-1 deficient mice have a normal response to thymus-dependent antigens, but have a reduced response to both type I and type II thymus independent antigens. However, lymphocyte populations in the spleen and peritoneum were not altered and no changes were observed in splenic architecture. Caveolin-1 deficient B-lymphocytes did not display altered proliferation in response to different stimuli. However, we found that Cav-1 deficient B cells have reduced IgG(3) secretion in vitro in response to LPS. Finally, we also demonstrate that human plasma cells (mature B lymphocytes) express Cav-1 in vivo. Taken, together these results provide convincing evidence for the expression of Cav-1 in activated B-lymphocytes and demonstrate a role for Cav-1 in the development of thymus-independent immune responses.