Peter van Swieten

Monoclonal antibodies to the major feline allergen Fel d I: I. Serologic and biologic activity of affinity-purified Fel d I and of Fel d I-depleted extract

Monoclonal antibodies were raised against a major feline allergen, Fel d I. The specificity of the antibodies for Fel d I was demonstrated by a modified crossed immunoelectrophoretic procedure. These antibodies were used in affinity purification and depletion. Fed d I was eluted from the affinity matrix at a low pH. It was tested in serologic and biologic assays. Results of both the RAST and the histamine release test with affinity-purified Fel d I confirmed that it is a very potent allergen for the majority of patients allergic to cats. In the extract depleted with the monoclonal antibody, the ratio Fel d I/cat albumin was reduced by a factor of 20. With this depleted extract, 18 of 25 patients showed a reduction greater than 50% of the bound anti-IgE in the RAST compared with the RAST with crude cat extract. In the histamine release test with cells from two patients, the activity of the depleted extract was reduced by factors of 30 and 40, respectively. Depletion of Fel d I by polyclonal antibodies resulted in a reduction of the ratio Fel d I/cat albumin by a factor of 330. In the RAST, a reduction of the IgE response greater than 50% was found in 23 of 25 patients; in 12 patients the reduction was greater than 90%. In the histamine release test, the polyclonally depleted extract was 200 to 300 times less potent than the crude cat-dander extract. These results indicate that a very large proportion of the allergenic activity of cat-dander extract is caused by a single molecule: Fel d I.

Viral Replication Capacity as a Correlate of HLA B57/B5801-Associated Nonprogressive HIV-1 Infection

HLA B57 and the closely related HLA B5801 are over-represented among HIV-1 infected long-term nonprogressors (LTNPs). It has been suggested that this association between HLA B57/5801 and asymptomatic survival is a consequence of strong CTL responses against epitopes in the viral Gag protein. Moreover, CTL escape mutations in Gag would coincide with viral attenuation, resulting in low viral load despite evasion from immune control. In this study we compared HLA B57/5801 HIV-1 infected progressors and LTNPs for sequence variation in four dominant epitopes in Gag and their ability to generate CTL responses against these epitopes and the autologous escape variants. Prevalence and appearance of escape mutations in Gag epitopes and potential compensatory mutations were similar in HLA B57/5801 LTNPs and progressors. Both groups were also indistinguishable in the magnitude of CD8+ IFN-γ responses directed against the wild-type or autologous escape mutant Gag epitopes in IFN-γ ELISPOT analysis. Interestingly, HIV-1 variants from HLA B57/5801 LTNPs had much lower replication capacity than the viruses from HLA B57/5801 progressors, which did not correlate with specific mutations in Gag. In conclusion, the different clinical course of HLA B57/5801 LTNPs and progressors was not associated with differences in CTL escape mutations or CTL activity against epitopes in Gag but rather with differences in HIV-1 replication capacity.

Highly active antiretroviral therapy with or without mycophenolate mofetil in treatment-naive HIV-1 patients

Objective: To study the effect of mycophenolate mofetil (MMF) on the decay rate of plasma HIV-1 RNA and the latently infected cellular reservoir in treatment-naive patients starting antiretroviral therapy. Design: Randomized trial. Methods: A group of 19 HIV-1 infected patients (9 with a chronic and 10 with a primary infection) starting a triple antiretroviral drug regimen were randomized to a group with or without MMF. Plasma samples for HIV-1 RNA were taken and HLA-DR−CD4+ T cells were co-cultured for HIV-1 isolation. Slopes of plasma HIV-1 RNA and cellular viral load decay were calculated for the first 14 days and the first 24 weeks of treatment, respectively. Results: The median plasma HIV-1 RNA daily decay rate in chronically infected patients was 0.25 log10 copies/ml [interquartile range (IQR), 0.18–0.30] with MMF and 0.28 log10 copies/ml (IQR, 0.22–0.32) without MMF (P = 0.56); in primary infected patients, it was 0.31 log10 copies/ml (IQR, 0.31–0.32) with MMF and 0.32 log10 copies/ml (IQR, 0.26–0.34) without MMF (P = 0.75). The median daily decay rate of latently infected cells was 0.017 and 0.004 infected cells/106 cells in patients with and without MMF, respectively (P = 0.89). The increase in CD4 T cells was comparable between patients with and without MMF. After stopping MMF, there was an increase in the cellular reservoir in six of eight patients. Conclusion: The addition of MMF to a triple class antiretroviral regimen in treatment-naive patients does not significantly increase the plasma HIV-1 RNA decay rate or the decay rate of the latently infected cellular reservoir.

Molecular Evolution of Human Immunodeficiency Virus Type 1 upon Transmission between Human Leukocyte Antigen Disparate Donor-Recipient Pairs

Background To address evolution of HIV-1 after transmission, we studied sequence dynamics in and outside predicted epitopes of cytotoxic T lymphocytes (CTL) in subtype B HIV-1 variants that were isolated from 5 therapy-naive horizontal HLA-disparate donor-recipient pairs from the Amsterdam Cohort Studies on HIV-1 infection and AIDS. Methodology/Principal Findings In the first weeks after transmission, the majority of donor-derived mutations in and outside donor-HLA-restricted epitopes in Gag, Env, and Nef, were preserved in the recipient. Reversion to the HIV-1 subtype B consensus sequence of mutations in- and outside donor-HLA-restricted CTL epitopes, and new mutations away from the consensus B sequence mostly within recipient-HLA-restricted epitopes, contributed equally to the early sequence changes. In the subsequent period (1–2 years) after transmission, still only a low number of both reverting and forward mutations had occurred. During subsequent long-term follow-up, sequence dynamics were dominated by forward mutations, mostly (50–85%) in recipient-HLA-restricted CTL epitopes. At the end of long-term follow-up, on average 43% of the transmitted CTL escape mutations in donor-HLA-restricted epitopes had reverted to the subtype B consensus sequence. Conclusions/Significance The relatively high proportion of long-term preserved mutations after transmission points to a lack of back selection even in the absence of CTL pressure, which may lead to an accumulating loss of critical CTL epitopes. Our data are supportive for a continuous adaptation of HIV-1 to host immune pressures which may have implications for vaccine design.

Differences between specificities of IgE and IgG4 antibodies: studies using recombinant chain 1 and chain 2 of the major cat allergen Felis domesticus (Fel d) I

IgE‐ and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE‐ and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE‐ and IgG4 binding was found. The fraction of Pel d I‐specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.

IgE and IgG4 binding to the ocelot variant of the cat (Felis domesticus) major allergen Fel d l

The cross-reactivity of human IgE and IgG4 antibodies of 22 cat-allergic patients with the ocelot variant of Fel d I was studied. Throughout the Felidae species, this allergen, found in cat saliva, dander, sebaceous glands, and lacrimal glands (1-3, 5) is conserved, as shown in radioallergosorbent test (RAST) inhibition assays with affinity-purified Fel d I and hair extracts from various members of the Felidae family (6). The Felidae species we used in these experiments originate from different lineages (4). Because of the phylogenetic distance among these species, they are useful tools to study the specificities of IgE and IgG4 responses. Other feline species, e.g., tiger and lion, were found to be so closely related as to be almost immunologically identical, and therefore less suited for this type of investigation.