R W Milne

International Federation of Clinical Chemistry (IFCC): Scientific Division, Committee on Apolipoproteins; Working Group of Antibody Reagents: Selection and Characterization of Monoclonal Antibodies for Measuring Plasma Levels of Apolipoproteins A-I and B

Measurements of apolipoproteins A-I and B are of growing interest in the clinical and research laboratory because levels of these proteins in the blood have been shown to serve as important predictors of atherosclerotic vascular disease, and they provide information not available from total cholesterol or lipoprotein cholesterol levels. Apolipoprotein A-I and B measurements have not reached their full potential in the clinical laboratory because of inadequate standardization and problems in methodology. Although both polyclonal and monoclonal antibodies have been used extensively in apolipoprotein assays, monoclonal antibodies offer the advantages of being chemically uniform, providing high specificity, and they can be produced in large amounts. As a result, more and more researchers and manufacturers have turned to monoclonal antibodies for the development of apolipoprotein immunoassays. However, generating and selecting monoclonal antibodies that are appropriate for the measurement of apolipoproteins A-I and B are difficult and time-consuming. On the other hand, an inadequate characterization of the monoclonal antibodies can lead to technical artefacts and to no comparability ofthe data. In order to facilitate the appropriate use of monoclonal antibodies in the measurement of apo A-I and B, some important considerations for generating and selecting suitable monoclonal antibodies have been outlined by the IFCC’s Committee on Apolipoproteins.

Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007-2013.

BACKGROUND US guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA, USA. METHODS Persons with primary HIV-1 infection have enrolled in an observational cohort at the University of Washington Primary Infection Clinic since 1992. We performed a retrospective analysis of plasma specimens collected prospectively from the 82 antiretroviral-naive subjects who were enrolled from 2007-2013, after FDA-approval of the first INSTI. Resistance testing was performed by consensus sequencing. RESULTS Specimens for analysis had been obtained a median of 24 (IQR 18-41, range 8-108) days after the estimated date of HIV-1 infection. All subjects were infected with HIV-1 subtype B except for one subject infected with subtype C. Consensus sequencing identified no subjects with major INSTI mutations (T66I, E92Q, G140S, Y143C/H/R, S147G, Q148H/K/R, N155H). Using exact binomial CIs, the upper bound of the 95% CI was 4.4%. CONCLUSIONS Although our sample size was small, this study does not support the need at this time to evaluate integrase mutations as part of routine consensus sequencing among persons newly diagnosed with HIV-1 infection. However, it is likely that the prevalence of transmitted INSTI mutations may increase with the recent commercial introduction of additional INSTIs and presumably greater INSTI use among persons living with HIV-1.

Transmission of HIV-1 drug resistance mutations within partner-pairs: A cross-sectional study of a primary HIV infection cohort

Background Transmission of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations, particularly that of minority drug-resistant variants, remains poorly understood. Population-based studies suggest that drug-resistant HIV-1 is less transmissible than drug-susceptible viruses. We compared HIV-1 drug-resistant genotypes among partner-pairs in order to assess the likelihood of transmission of drug resistance mutations and investigate the role of minority variants in HIV transmission. Methods and findings From 1992–2010, 340 persons with primary HIV-1 infection and their partners were enrolled into observational research studies at the University of Washington Primary Infection Clinic (UWPIC). Out of 50 partner-pairs enrolled, 36 (72%) transmission relationships were confirmed by phylogenetic distance analysis of HIV-1 envelope (env) sequences, and 31 partner-pairs enrolled after 1995 met criteria for this study. Drug resistance mutations in the region of the HIV-1 polymerase gene (pol) that encodes protease and reverse transcriptase were assessed by 454-pyrosequencing. In 25 partner-pairs where the transmission direction could be determined, 12 (48%) transmitters had 1–4 drug resistance mutations (23 total) detected in their HIV-1 populations at a median frequency of 6.0% (IQR 1.5%–98.7%, range 1.0%–99.6%). Of 10 major mutations detected in five transmitters at a frequency >95%, 100% (95% CI 69.2%–100%) were detected in recipients. All of these transmitters were antiretroviral (ARV)-naïve at the time of specimen collection. Fourteen mutations (eight major mutations and six accessory mutations) were detected in nine transmitters at low frequencies (1.0%–11.8%); four of these transmitters had previously received ARV therapy. Two (14% [95% CI 1.8%–42.8%]) G73S accessory mutations were detected in both transmitter and recipient. This number is not significantly different from the number expected based on the observed frequencies of drug-resistant viruses in transmitting partners. Limitations of this study include the small sample size and uncertainties in determining the timing of virus transmission and mutation history. Conclusions Drug-resistant majority variants appeared to be commonly transmitted by ARV-naïve participants in our analysis and may contribute significantly to transmitted drug resistance on a population level. When present at low frequency, no major mutation was observed to be shared between partner-pairs; identification of accessory mutations shared within a pair could be due to transmission, laboratory artifact, or apolipoprotein B mRNA-editing enzyme, catalytic polypeptides (APOBECs), and warrants further study.