Huan H. Nguyen

Heterosubtypic Immunity to Influenza A Virus Infection Requires B Cells but Not CD8+ Cytotoxic T Lymphocytes

Heterosubtypic immunity (HSI), defined as protective cross-reactivity to lethal infection with influenza A virus of a serotype different from the virus initially encountered, is thought to be mediated by cross-reactive cytotoxic T lymphocytes (CTL). This study provides direct evidence for the role of effector CTL versus B cells in HSI in mice with a targeted disruption in the alpha chain of CD8 molecule (CD8(+) T cell deficient) or the immunoglobulin mu heavy chain (B cell deficient), respectively. CD8(+) T cell-deficient mice developed complete HSI. These mice displayed normal humoral immune responses, as determined by titers of subtype cross-reactive antibodies and virus-neutralizing antibodies specific for the immunizing influenza strain. In contrast, HSI was not observed in B cell-deficient mice, although these mice could mount cross-reactive CTL responses. These results show that B cells are required for HSI and provide new insight into the mechanisms of HSI, with significant implications in vaccine development.

Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity.

Gamma Interferon Is Not Required for Mucosal Cytotoxic T-Lymphocyte Responses or Heterosubtypic Immunity to Influenza A Virus Infection in Mice

ABSTRACT Heterosubtypic immunity (HSI) is defined as cross-protection against influenza virus of a different serotype than the virus initially encountered and is thought to be mediated by influenza virus-specific cytotoxic T lymphocytes (CTL). Since gamma interferon (IFN-γ) stimulates cytotoxic cells, including antigen-specific CTL which may control virus replication by secretion of antiviral cytokines such as tumor necrosis factor alpha and IFN-γ, we have investigated the mechanism of HSI by analyzing the role of IFN-γ for HSI in IFN-γ gene-deleted (IFN-γ−/−) mice. It has been reported that IFN-γ is not required for recovery from primary infection with influenza virus but is important for HSI. Here, we conclusively show that IFN-γ is not required for induction of secondary influenza virus-specific CTL responses in mediastinal lymph nodes and HSI to lethal influenza A virus infection. Although T helper 2 (Th2)-type cytokines were upregulated in the lungs of IFN-γ−/− mice after virus challenge, either Th1- or Th2-biased responses could provide heterosubtypic protection. Furthermore, titers of serum-neutralizing and cross-reactive antibodies to conserved nucleoprotein in IFN-γ−/− mice did not differ significantly from those in immunocompetent mice. These results indicate that lack of IFN-γ does not impair cross-reactive virus-specific immune responses and HSI to lethal infection with influenza virus. Our findings provide new insight for the mechanisms of HSI and should be valuable in the development of protective mucosal vaccines against variant virus strains, such as influenza and human immunodeficiency virus.

Mechanism of heterosubtypic immunity to influenza A virus infection

Abstract Heterosubtypic immunity (HSI) is defined as protective cross-reactive immune responses to lethal infection with influenza A virus of a different serotype than the virus initially encountered, and is thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL). These CTL recognize conserved epitopes of internal proteins, such as nucleoprotein (NP) or matrix (M) protein shared by influenza A virus subtypes. Despite extensive studies, the precise effector mechanism for HSI remains elusive. For example, our recent studies and those of others reported HSI in T cell-depleted, β2-microglobulin-deficient, and CD8 cell-deficient mice. The role for humoral immune responses in HSI is also unclear. Passive transfer of heterosubtypic immune serum did not provide protection against lethal heterosubtypic challenge, while B cell-deficient mice failed to develop HSI. Our recent findings and those of others now allow us to suggest a two-tiered HSI. Early after heterosubtypic challenge, a number of factors including subtype-specific CTL as well as antibody (Ab) responses and other as yet not well characterized host factors are able to minimize temporarily the virus spread, but are unable to clear the infection. In the later phase, the development of virus-neutralizing (VN) antibodies is important for virus clearance resulting in complete host recovery.

Analysis of influenza specific CTL responses in children using fibroblasts as stimulator and target cells

Abstract Determination of cytotoxic T lymphocyte (CTL) responses in humans by traditional assays have been restricted by the volumes of blood needed and the nature of the target cells used for the assays. A number of technological advances have recently been described which improve quantitation and sensitivity of the assays. We are developing the concept of using fibroblasts as permissive targets for respiratory viruses in CTL assays. Methods: Fibroblasts can be grown from dispersed nasal epithelium obtained from adenoids. These cells infected with influenza are used as a stimulator layer for lymphocytes. They also can form the target for CTLs in a chromium release assay. Results: Comparison showed fibroblasts to be comparable to influenza infected lymphocytes as stimulators and to EBV transformed lymphocytes as targets. Cells from 25 adenoids have been explored in the fibroblast CTL system with about half of the cryoperserved peripheral blood lymphocytes showing CTL activity and mucosally associated CTLs being found in one patient. Conclusions: Exploration of new CTL targets that may be adapted not only to chromium release assays but to newer CTL assays offers the promise of developing a practical way of defining the CTL response in young children to natural infection and to varying immunization strategies. This may allow us to put in perspective CTLs as a correlate of immunity to influenza in humans.