Longevity and Mechanism of Heterosubtypic Protection Induced by M2SR (M2-Deficient Single-Replication) Live Influenza Virus Vaccine in Mice.

Seasonal influenza and the threat of global pandemics present a continuing threat to public health. However, conventional inactivated influenza vaccines (IAVs) provide little cross-protective immunity and suboptimal efficacy, even against well-matched strains. Furthermore, the protection against matched strains has been shown to be of a short duration in both mouse models and humans. M2SR (M2-deficient single-replication influenza virus) is a single-replication vaccine that has been shown to provide effective cross-protection against heterosubtypic influenza viruses in both mouse and ferret models. In the present study, we investigated the duration and mechanism of heterosubtypic protection induced by M2SR in a mouse model. We previously showed that M2SR generated from influenza A/Puerto Rico/8/34 (H1N1) significantly protected C57BL/6 mice against lethal challenge with both influenza A/Puerto Rico/8/34 (H1N1, homosubtypic) and influenza A/Aichi/2/1968 (H3N2, heterosubtypic), whereas the inactivated influenza vaccine provided no heterosubtypic protection. The homosubtypic protection induced by M2SR was robust and lasted for greater than 1 year, whereas that provided by the inactivated vaccine lasted for less than 6 months. The heterosubtypic protection induced by M2SR was of a somewhat shorter duration than the homosubtypic protection, with protection being evident 9 months after vaccination. However, heterosubtypic protection was not observed at 14 months post vaccination. M2SR has been shown to induce strong systemic and mucosal antibody and T cell responses. We investigated the relative importance of these immune mechanisms in heterosubtypic protection, using mice that were deficient in B cells or mice that were depleted of T cells immediately before challenge. Somewhat surprisingly, the heterosubtypic protection was completely dependent on B cells in this model, whereas the depletion of T cells had no significant effect on survival after a lethal heterosubtypic challenge. While antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated to be important in the response to some influenza vaccines, a lack of Fc receptors did not affect the survival of M2SR-vaccinated mice following a lethal challenge. We examined the influenza proteins targeted by the heterosubtypic antibody response. Shortly after the H1N1 M2SR vaccination, high titers of cross-reactive antibodies to heterosubtypic H3N2 nucleoprotein (NP) and lower titers to the stalk region of the hemagglutinin (HA2) and neuraminidase (NA) proteins were observed. The high antibody titers to heterosubtypic NP persisted one year after vaccination, whereas the antibody titers to the heterosubtypic HA2 and NA proteins were very low, or below the limit of detection, at this time. These results show that the intranasal M2SR vaccine elicits durable protective immune responses against homotypic and heterosubtypic influenza infection not seen with intramuscular inactivated vaccines. Both the homo- and heterosubtypic protection induced by the single-replication vaccine are dependent on B cells in this model. While the homosubtypic protection is mediated by antibodies to the head region of HA, our data suggest that the heterosubtypic protection for M2SR is due to cross-reactive antibodies elicited against the NP, HA2, and NA antigens that are not targeted by current seasonal influenza vaccines.

Repercussions of the COVID-19 pandemic on preventive health services in Brazil.

INTRODUCTION: The increasing burden of non-communicable diseases and limited public financing are major challenges facing health care systems in Latin America. Although COVID-19 severely impacted the Brazilian health care system, it is crucial to further characterize the degree of disruption caused to public health efforts, in order to address and manage long term effects of this pandemic. We therefore quantified the demand for preventive and treatment services from the Brazilian Unified Health System (Sistema Único de Saúde/SUS) in 2020 to evaluate potential repercussions of COVID-19 in this setting. METHODS: Using the SUS database, we compared preventative and treatment services rendered in 2020 to the same services rendered from 2017 to 19. We also evaluated the frequency of respiratory infection (RI) diagnoses during the pandemic, relative to the preceding years. RESULTS: Compared to 2017-19, in 2020 non-urgent medical appointments decreased 1.4-fold (p = 0.0017), dental consultations 2.8-fold (p = 0.05), and immunization coverage 1.5 fold (p = 0.0005). The number of RI visits to SUS ambulatory care units in 2020 was 4.2 times higher than in preceding years (p = 0.0014), with a peak of 280,898 diagnoses in July 2020. CONCLUSION: The COVID-19 pandemic appears to have led to a dramatic decline in preventative and treatment services provided by SUS to the Brazilian population. Our findings may aid decision-makers in formulating policies to increase the availability of outpatient services in the aftermath of the pandemic. Counter measures will be critical to avoid a resurgence in vaccine-preventable diseases and complications stemming from non-communicable, chronic health conditions.

Post-acute COVID-19 syndrome after reinfection and vaccine breakthrough by the SARS-CoV-2 Gamma variant in Brazil.

We describe a case of prolonged COVID-19 caused by the SARS-CoV-2 Gamma variant in a fully vaccinated healthcare worker, 387 days after an infection caused by lineage B.1.1.33. Infections were confirmed by whole-genome sequencing and corroborated by the detection of neutralizing antibodies in convalescent serum samples. Considering the permanent exposure of this healthcare worker to SARS-CoV-2, the waning immunity after the first infection, the low efficacy of the inactivated vaccine at preventing COVID-19, the immune escape of the Gamma variant (VOC), and the burden of post-COVID syndrome, this individual would have benefited from an additional dose of a heterologous vaccine.

ZIKA Virus Neutralizing Antibody Kinetics in Antenatally Exposed Infants.

BACKGROUND: Zika virus (ZIKV) is associated with severe congenital abnormalities and laboratory diagnosis of antenatal infection is difficult. Here we evaluated ZIKV neutralizing antibody (nAb) kinetics in infants born to mothers with PCR-confirmed ZIKV infection during pregnancy. METHODS: Neonates (n = 98) had serum specimens tested repeatedly for ZIKV nAb over the first 2 years of life using virus neutralization test (VNT). ZIKV neonatal infection was confirmed by RT-PCR in blood or urine and/or presence of ZIKV IgM antibodies, and results were correlated with infant clinical features. RESULTS: Postnatal laboratory evidence of ZIKV vertical transmission was obtained for 60.2% of children, while 32.7% exhibited clinical abnormalities. Congenital abnormalities were found in 37.3% of children with confirmed ZIKV infection and 31.0% of children without confirmed infection (P = .734). All but 1 child displayed a physiologic decline in ZIKV nAb, reflecting maternal antibody decay, despite an early ZIKV-IgM response in one-third of infants. CONCLUSIONS: Infants with antenatal ZIKV exposure do not develop ZIKV nAb despite an early IgM response. Therefore, ZIKV VNT in children is not useful for diagnosis of congenital infection. In light of these findings, it remains to be determined if children infected in utero are potentially susceptible to reinfection.

Zika virus vertical transmission in children with confirmed antenatal exposure.

We report Zika virus (ZIKV) vertical transmission in 130 infants born to PCR+ mothers at the time of the Rio de Janeiro epidemic of 2015-2016. Serum and urine collected from birth through the first year of life were tested by quantitative reverse transcriptase polymerase chain reaction (PCR) and/or IgM Zika MAC-ELISA. Four hundred and seven specimens are evaluated; 161 sera tested by PCR and IgM assays, 85 urines by PCR. Sixty-five percent of children (N = 84) are positive in at least one assay. Of 94 children tested within 3 months of age, 70% are positive. Positivity declines to 33% after 3 months. Five children are PCR+ beyond 200 days of life. Concordance between IgM and PCR results is 52%, sensitivity 65%, specificity 40% (positive PCR results as gold standard). IgM and serum PCR are 61% concordant; serum and urine PCR 55%. Most children (65%) are clinically normal. Equal numbers of children with abnormal findings (29 of 45, 64%) and normal findings (55 of 85, 65%) have positive results, p = 0.98. Earlier maternal trimester of infection is associated with positive results (p = 0.04) but not clinical disease (p = 0.98). ZIKV vertical transmission is frequent but laboratory confirmed infection is not necessarily associated with infant abnormalities.

The Potential for Immunospecific Therapy in Multiple Sclerosis Based on Identification of Driver Clones of the Disease.

The autoimmune disease multiple sclerosis (MS) is driven by T cells that are reactive to self-antigens of the brain and spinal cord. Many drugs have been developed to treat MS, but we believe that immune-specific targeting of pathogenic T cells may be a better approach for treatment. This type of therapy identifies specific components of the self-reactive T-cell repertoire that would undergo similar natural selection criteria as those found in driver genes in cancer genesis. In the context of autoimmunity, we propose that a focused subpopulation of T cells "drive" disease and could be found in higher frequency and become over-represented during disease induction and subsequent MS relapses. In addition, identification of other key signatures of driver T cells is important. One such marker could be interleukin (IL)-17- producing T cells. Here, we discuss the use of experimental autoimmune encephalomyelitis (EAE) animal models (that mimic many pathologic mechanisms involved in MS) to identify possible driver clones of this autoimmunity within the set of T cells expressing the IL-17 cytokine. EAE can be induced by myelin injection-associated proteins in adjuvants. The disease model in the Swiss/Jackson laboratory mouse strain represents the most common form of MS in humans: relapsing remitting MS. Finally, we discuss the concept of using IL-17 as a marker for pathogenic T cells, combined with identifying their T-cell receptor V repertoire, which could provide targeted approaches designed to neutralize driver T cells for MS immunotherapy.

Single-replication BM2SR vaccine provides sterilizing immunity and cross-lineage influenza B virus protection in mice.

Both influenza A and B viruses cause outbreaks of seasonal influenza resulting in significant morbidity and mortality. There are two antigenically distinct lineages of influenza B virus, Yamagata lineage (YL) and Victoria lineage (VL). Since both B lineages have been co-circulating for years, more than 70% of influenza vaccines currently manufactured are quadrivalent consisting of influenza A (H1N1), influenza A (H3N2), influenza B (YL) and influenza B (VL) antigens. Although quadrivalent influenza vaccines tend to elevate immunity to both influenza B lineages, estimated overall vaccine efficacy against influenza B is still only around 42%. Thus, a more effective influenza B vaccine is needed. To meet this need, we generated BM2-deficient, single-replication (BM2SR) influenza B vaccine viruses that encode surface antigens from influenza B/Wisconsin/01/2010 (B/WI01, YL) and B/Brisbane/60/2008 (B/Bris60, VL) viruses. The BM2SR-WI01 and BM2SR-Bris60 vaccine viruses are replication-deficient in vitro and in vivo, and can only replicate in a cell line that expresses the complementing BM2 protein. Both BM2SR viruses were non-pathogenic to mice, and vaccinated animals showed elevated mucosal and serum antibody responses to both Yamagata and Victoria lineages in addition to cellular responses. Serum antibody responses included lineage-specific hemagglutinin inhibition antibody (HAI) responses as well as responses to the stem region of the hemagglutinin (HA). BM2SR vaccine viruses provided apparent sterilizing immunity to mice against intra- and inter-lineage drifted B virus challenge. The data presented here support the feasibility of BM2SR as a platform for next-generation trivalent influenza vaccine development.

Delayed childhood neurodevelopment and neurosensory alterations in the second year of life in a prospective cohort of ZIKV-exposed children.

We report neurodevelopmental outcomes in 216 infants followed since the time of PCR-confirmed maternal Zika virus (ZIKV) infection in pregnancy during the Rio de Janeiro epidemic of 2015-2016 (refs. 1,2). Neurodevelopment was assessed by Bayley Scales of Infant and Toddler Development, third edition (Bayley-III; cognitive, language and motor domains) in 146 children and through neurodevelopment questionnaires/neurological examinations in 70 remaining children. Complete eye exams (n = 137) and hearing assessments (n = 114) were also performed. Below-average neurodevelopment and/or abnormal eye or hearing assessments were noted in 31.5% of children between 7 and 32 months of age. Among children assessed by Bayley-III, 12% scored below -2 s.d. (score <70; a score of 100 ± 2 s.d. is the range) in at least one domain; and 28% scored between -1 and -2 s.d. in any domain (scores <85-70). Language function was most affected, with 35% of 146 children below average. Improved neurodevelopmental outcomes were noted in female children, term babies, children with normal eye exams and maternal infection later in pregnancy (P = 0.01). We noted resolution of microcephaly with normal neurodevelopment in two of eight children, development of secondary microcephaly in two other children and autism spectrum disorder in three previously healthy children in the second year of life.

Behavioral, climatic, and environmental risk factors for Zika and Chikungunya virus infections in Rio de Janeiro, Brazil, 2015-16.

The burden of arboviruses in the Americas is high and may result in long-term sequelae with infants disabled by Zika virus infection (ZIKV) and arthritis caused by infection with Chikungunya virus (CHIKV). We aimed to identify environmental drivers of arbovirus epidemics to predict where the next epidemics will occur and prioritize municipalities for vector control and eventual vaccination. We screened sera and urine samples (n = 10,459) from residents of 48 municipalities in the state of Rio de Janeiro for CHIKV, dengue virus (DENV), and ZIKV by molecular PCR diagnostics. Further, we assessed the spatial pattern of arbovirus incidence at the municipal and neighborhood scales and the timing of epidemics and major rainfall events. Lab-confirmed cases included 1,717 infections with ZIKV (43.8%) and 2,170 with CHIKV (55.4%) and only 29 (<1%) with DENV. ZIKV incidence was greater in neighborhoods with little access to municipal water infrastructure (r = -0.47, p = 1.2x10-8). CHIKV incidence was weakly correlated with urbanization (r = 0.2, p = 0.02). Rains began in October 2015 and were followed one month later by the largest wave of ZIKV epidemic. ZIKV cases markedly declined in February 2016, which coincided with the start of a CHIKV outbreak. Rainfall predicted ZIKV and CHIKV with a lead time of 3 weeks each time. The association between rainfall and epidemics reflects vector ecology as the larval stages of Aedes aegypti require pools of water to develop. The temporal dynamics of ZIKV and CHIKV may be explained by the shorter incubation period of the viruses in the mosquito vector; 2 days for CHIKV versus 10 days for ZIKV.

Maternal Zika Virus Disease Severity, Virus Load, Prior Dengue Antibodies, and Their Relationship to Birth Outcomes.

BACKGROUND: Congenital Zika virus (ZIKV) syndrome is a newly identified condition resulting from infection during pregnancy. We analyzed outcome data from a mother-infant cohort in Rio de Janeiro in order to assess whether clinical severity of maternal ZIKV infection was associated with maternal virus load, prior dengue antibodies, or abnormal pregnancy/infant outcomes. METHODS: A clinical severity assessment tool was developed based on duration of fever, severity of rash, multisystem involvement, and duration of symptoms during ZIKV infection. ZIKV-RNA load was quantified by polymerase chain reaction (PCR) cycles in blood/ urine. Dengue immunoglobulin G (IgG) antibodies were measured at baseline. Adverse outcomes were defined as fetal loss or a live infant with grossly abnormal clinical or brain imaging findings. Regression models were used to study potential associations. RESULTS: 131 ZIKV-PCR positive pregnant women were scored for clinical disease severity, 6 (4.6%) had mild disease, 98 (74.8%) had moderate disease, and 27 (20.6%) severe manifestations of ZIKV infection. There were 58 (46.4%) abnormal outcomes with 9 fetal losses (7.2%) in 125 pregnancies. No associations were found between: disease severity and abnormal outcomes (P = .961; odds ratio [OR]: 1.00; 95% confidence interval [CI]: 0.796-1.270); disease severity and viral load (P = .994); viral load and adverse outcomes (P = .667; OR: 1.02; 95% CI: 0.922-1.135); or existence of prior dengue antibodies (88% subjects) with severity score, ZIKV-RNA load or adverse outcomes (P = .667; OR: 0.78; 95% CI: 0.255-2.397). CONCLUSIONS: Congenital ZIKV syndrome does not appear to be associated with maternal disease severity, ZIKV-RNA load at time of infection or existence of prior dengue antibodies.

Zika virus and diagnostics.

PURPOSE OF REVIEW: The purpose of this review is to present what is known about the Zika virus (ZIKV) at the time of writing this review. The viral structure and its phylogeny, as well as the limitations of current available techniques used for diagnosis, are discussed. RECENT FINDINGS: Crystallography and cryo-electron microscopy of the whole ZIKV, or a few of its proteins, are confirming its overall antigenic relatedness to other flaviviruses. Sequencing has revealed its dynamic genetic variation and has placed the Western cluster of Zika isolates within the Asian phylogenic tree. Genetic codon mutations, although highly prevalent, do not usually translate into modifications at amino acid or proteomic levels, revealing conserved enzymatic functions that could potentially be addressed therapeutically. Clinical characterization of ZIKV infection is complicated because of symptoms similar to dengue and chikungunya. Diagnosis requires specialized laboratories with costly reagents and highly trained personnel. Although commercial labs are now offering ZIKV diagnostic tests, most of them are not fully tested in comparison with standard molecular techniques standardized at CDC and local health departments. We are still in desperate need of simpler diagnostic tests that better discriminate ZIKV from coendemic arboviruses. SUMMARY: The area of better Zika diagnostic assays is a rapidly developing field with the public attention directed to this epidemic. Academic interest in this topic is driving fast disclosure of information in peer-reviewed journals and grey papers via web-based forums. We expect in the near future that new promising strategies for improved Zika diagnostics will translate into preventive and therapeutic tools.

Zika Virus Infection in Pregnant Women in Rio de Janeiro.

BACKGROUND: Zika virus (ZIKV) has been linked to central nervous system malformations in fetuses. To characterize the spectrum of ZIKV disease in pregnant women and infants, we followed patients in Rio de Janeiro to describe clinical manifestations in mothers and repercussions of acute ZIKV infection in infants. METHODS: We enrolled pregnant women in whom a rash had developed within the previous 5 days and tested blood and urine specimens for ZIKV by reverse-transcriptase-polymerase-chain-reaction assays. We followed women prospectively to obtain data on pregnancy and infant outcomes. RESULTS: A total of 345 women were enrolled from September 2015 through May 2016; of these, 182 women (53%) tested positive for ZIKV in blood, urine, or both. The timing of acute ZIKV infection ranged from 6 to 39 weeks of gestation. Predominant maternal clinical features included a pruritic descending macular or maculopapular rash, arthralgias, conjunctival injection, and headache; 27% had fever (short-term and low-grade). By July 2016, a total of 134 ZIKV-affected pregnancies and 73 ZIKV-unaffected pregnancies had reached completion, with outcomes known for 125 ZIKV-affected and 61 ZIKV-unaffected pregnancies. Infection with chikungunya virus was identified in 42% of women without ZIKV infection versus 3% of women with ZIKV infection (P<0.001). Rates of fetal death were 7% in both groups; overall adverse outcomes were 46% among offspring of ZIKV-positive women versus 11.5% among offspring of ZIKV-negative women (P<0.001). Among 117 live infants born to 116 ZIKV-positive women, 42% were found to have grossly abnormal clinical or brain imaging findings or both, including 4 infants with microcephaly. Adverse outcomes were noted regardless of the trimester during which the women were infected with ZIKV (55% of pregnancies had adverse outcomes after maternal infection in the first trimester, 52% after infection in the second trimester, and 29% after infection in the third trimester). CONCLUSIONS: Despite mild clinical symptoms in the mother, ZIKV infection during pregnancy is deleterious to the fetus and is associated with fetal death, fetal growth restriction, and a spectrum of central nervous system abnormalities. (Funded by Ministério da Saúde do Brasil and others.).

GM-CSF production allows the identification of immunoprevalent antigens recognized by human CD4+ T cells following smallpox vaccination.

The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a "T cell-driven" methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igµ(null) mice after polyclonal B cell reconstitution.

BACKGROUND: Non Obese Diabetic mice lacking B cells (NOD.Igµ(null) mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age. METHODS: We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igµ(null) mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes. RESULTS: We found that B cell reconstitution of NOD.Igµ(null) mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igµ(null) mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro. CONCLUSIONS: Diabetes in NOD.Igµ(null) mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.

Dietary aspartyl-phenylalanine-1-methyl ester delays osteoarthritis and prevents associated bone loss in STR/ORT mice.

OBJECTIVE: STR/ORT mice provide a well-known model for murine idiopathic OA, with histological joint lesions resembling those of human OA. This model was used to investigate protective effects of the dipeptide aspartyl-phenylalanine-1-methyl ester (Asp-Phe-OMe or aspartame) via the oral route vs a regular diet. METHODS: STR/ORT mice were housed individually and fed diets with or without Asp-Phe-OMe (4 mg/kg), after weaning at the age of 3 weeks, until 15 months of age (average of 20 animals per group). The study groups were kept blinded to the investigators, who measured food consumption and body weight and performed gait mobility tests. Radiographic scans were also performed at regular time intervals to evaluate differential radiographic anomalies associated with progress of OA in response to oral Asp-Phe-OMe therapy. RESULTS: The Asp-Phe-OMe-fed animals presented a pattern of significantly delayed disease onset. In addition, their muscle and bone mass were highly preserved, even at later time points after OA was established. Moreover, control animals presented a higher variability in gait motility in comparison with the Asp-Phe-OMe-fed animals, suggesting a protective effect from movement limitations associated with advanced OA. CONCLUSION: Asp-Phe-OMe, given orally, delays OA in the spontaneous STR/ORT model, improves bone cortical density and muscle mass, and may contribute to a better quality of life for these diseased animals.

Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice.

BACKGROUND: Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. METHODS: The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. RESULTS: Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. CONCLUSION: Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.

Altered peptide ligands can modify the Th2 T cell response to the immunodominant 161-175 peptide of LACK (Leishmania homolog for the receptor of activated C kinase).

Following Leishmania major infection, the early LACK (Leishmania homolog of receptors for activated C kinase)-induced IL-4 response appears to determine disease susceptibility in BALB/c mice. Therefore, we sought to manipulate the pathogenic T cell responses to the immunodominant epitope with the use of altered peptide ligands (APLs). Conservative and non-conservative substitutions for each amino acid of the LACK 161-175 peptide determinant were tested for their stimulatory capacity in four different LACK-reactive T cell systems. From these results, we propose a likely LACK 163-171/I-A(d) core peptide register and show that APLs with changes at putative T cell receptor (TCR) contacts provide the greatest potential for immune deviation. In particular, the TCR-contact H164V APL expanded Th1 cells upon in vitro recall of naïve splenocytes from LACK-specific BV4 T cell receptor transgenic mice and stimulated IFN-gamma secretion from a Th2-committed LACK-reactive T cell line. We also observed that non-conservative substitutions flanking the core determinant had strong agonistic effects for proliferation and Th1/Th2 modulation. However, upon immunization, the H164V APL considerably downregulated proliferation and cytokine responses to the wild type LACK 161-175 peptide, while immunization with the weak agonist, MHC contact APL S171K, increased the IFN-gamma/IL-4 ratio to the wild type peptide. In these instances, a hyporesponsive T cell response to the wild-type peptide was achieved by immunizing with an APL possessing non-conservative substitutions at TCR contact sites, while immune deviation was accomplished using a weak-agonist APL that retained the core determinant. Thus, certain LACK-APLs are able to induce T cell responses with a protective phenotype in an infectious disease such as leishmaniasis.