Stefan Kostense

HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors

It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.

Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity

ABSTRACT Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.

Immunogenicity of Recombinant Adenovirus Serotype 35 Vaccine in the Presence of Pre-Existing Anti-Ad5 Immunity

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4+ T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.

High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function.

To investigate the effect of HIV‐specific CD8+ T cells on viral plasma load and disease progression, we enumerated HLA‐A2‐, B8‐ and B57‐restricted CD8+ T cells directed against several HIV epitopes in a total of 54 patients by the use of tetrameric HLA‐peptide complexes. In patients with high CD4+ T cell numbers, HIV‐specific tetramer+ cells inversely correlated with viral load. Patients with CD4+ T cell numbers below 400/μ l blood, however, carried high viral load despite frequently having high tetramer+ T cell numbers. This lack of correlation between viral load and tetramer+ cells did not result from viral escape variants, as in only 4 of 13 patients, low frequencies of viruses with mutated epitopes were observed. In 15 patients we measured CD8+ T cell antigen responsiveness to HIV peptide stimulation in vitro. FACS analyses showed differential IFN‐γ production of the tetramer+ cells, and this proportion of IFN‐γ‐producing tetramer+ cells correlated with AIDS‐free survival and with T cell maturation to the CD27– effector stage. These data show that most HIV‐infected patients have sustained HIV‐specific T cell expansions but many of these cells seem not to be functional, leaving the patient with high numbers of non‐functional virus‐specific CD8+ T cells in the face of high viral burden.

Neutralizing Antibodies and CD8+ T Lymphocytes both Contribute to Immunity to Adenovirus Serotype 5 Vaccine Vectors

ABSTRACT The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8+ T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8+ T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.

Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector.

The seroprevalence of adenovirus types 5 (Ad5) and 35 (Ad35) was investigated in patients at risk of AIDS. The seroprevalence of Ad5 was higher than Ad35 in HIV-infected patients from The Netherlands (60% versus 7%) and sub-Saharan Africa (90% versus 20%). The seroprevalence was similar among HIV-infected and uninfected individuals, and remained constant during progression to AIDS. Ad35 is less prone to neutralization than Ad5, encouraging the further development of Ad35 for vaccination against HIV.

Lack of Epstein-Barr virus- and HIV-specific CD27- CD8+ T cells is associated with progression to viral disease in HIV-infection

Objective: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein–Barr virus (EBV)-specific CD8+ T cells was performed. Design: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphomas (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally. Methods: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. Results: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27− T cells, in contrast to rising numbers of EBV-specific CD27− CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27− CD8+ T cells. Higher numbers of HIV-specific CD27− CD8+ T cells were associated with delayed disease progression. Virus-specific CD27− T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. Conclusions: Taken together, our data indicate that virus-specific CD27− T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27− effector T cells may lead to progression of viral disease.

Preclinical Models Used for Immunogenicity Prediction of Therapeutic Proteins

All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.

Induction of CAMEL/NY-ESO-ORF2-specific CD8+ T cells upon stimulation with dendritic cells infected with a modified Ad5 vector expressing a chimeric Ad5/35 fiber

Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8+ T cells specific for the known HLA-A*0201-binding CAMEL/NY-ESO-ORF21–11 epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8+ T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF246–54 epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8+ T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8+ T cell responses in vivo.

Validation of the Rapid Fluorescent Focus Inhibition Test for Rabies Virus-Neutralizing Antibodies in Clinical Samples

ABSTRACT Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.