Supplemental Material - HIV drug resistance in the era of contemporary antiretroviral therapy: A clinical perspective

Supplemental Material for HIV drug resistance in the era of contemporary antiretroviral therapy: A clinical perspective by Andrew Carr, Nicola E Mackie, Roger Paredes and Kiat Ruxrungtham.</p

(A) Dengue DNA vaccine construction.

<p>Humanized sequence of the consensus <i>prME</i> genes from each dengue serotypes were cloned into pCMVkan expression vector. ss: signal sequence; HCMV: human CMV promoter. (B) Intracellular dengue proteins expression. Vero cells were transfected with indicated dengue DNA vaccine constructs or infected with DENV. The transfected or infected cells were stained with DAPI, anti-flaviviruses E mAb (4G2) or anti-DENV NS1 antibody, and analyzed using fluorescence microscopy. (C) Immunoblot analysis of secreted E protein. Cell culture supernatants were collected at 24 hr post transfection or infection, and analyzed by employing anti-flavivirus E antibody (clone 4G2). Lanes 1–5, recombinant plasmid pCMVkanD1, −2, −3, −4 prME and pCMVkan empty vector; lane 6, DENV-2 strain 16681. M: protein marker.</p

NAb responses against reference viruses used in PRNT in mice immunized with TDNA.

<p>NAb activities in sera of individual mouse following an IM-EP immunization with 100 µg/dose (opened diamond) and 10 µg/dose (grey square) for three times were determined at week 4 after the third dose. Horizontal lines represent the median PRNT50 titer for each group of mice (<i>n</i> = 5–6). ns: no statistically significant difference.</p

Kinetics of NAb responses following TDNA prime-boost immunization.

<p>NAb titers of individual mice sera (<i>n</i> = 6), against each of the four dengue serotypes were shown separately. Opened circle and grey square represent the median PRNT50 titer with inter-quartile ranges for the TDNA group and the empty vector group, respectively. Arrows represent the injection of each dose of TDNA. * indicates <i>p</i><0.05.</p

Role of Rilpivirine and Etravirine in Efavirenz and Nevirapine-Based Regimens Failure in a Resource-Limited Country: A Cross- Sectional Study

<div><p>Introduction</p><p>Etravirine(ETR) can be used for patients who have failed NNRTI-based regimen. In Thailand, ETR is approximately 45 times more expensive than rilpivirine(RPV). However, there are no data of RPV use in NNRTI failure. Therefore, we assessed the susceptibility and mutation patterns of first line NNRTI failure and the possibility of using RPV compared to ETV in patients who have failed efavirenz(EFV)- and nevirapine(NVP)-based regimens.</p><p>Methods</p><p>Clinical samples with confirmed virological failure from EFV- or NVP-based regimens were retrospectively analyzed. Resistance-associated mutations (RAMs) were interpreted by IAS-USA Drug Resistance Mutations. Susceptibility of ETR and RPV were interpreted by DUET, Monogram scoring system, and Stanford University HIV Drug Resistance Database.</p><p>Results</p><p>1,279 and 528 patients failed EFV- and NVP-based regimens, respectively. Y181C was the most common NVP-associated RAM (54.3% vs. 14.7%, p<0.01). K103N was the most common EFV-associated RAM (56.5% vs. 19.1%, P<0.01). The results from all three scoring systems were concordant. 165(11.1%) and 161(10.9%) patients who failed NVP-based regimen were susceptible to ETR and RPV, respectively (p = 0.85). 195 (32.2%) and 191 (31.6%) patients who failed EFV-based regimen, were susceptible to ETR and RPV, respectively (p = 0.79). The susceptibility of ETV and RPV in EFV failure was significantly higher than NVP failure (p<0.01).</p><p>Conclusion</p><p>The mutation patterns for ETR and RPV were similar but 32% and 11% of patients who failed EFV and NVP -based regimen, respectivly were susceptible to RPV. This finding suggests that RPV can be used as the alternative antiretroviral agent in patients who have failed EFV-based regimen.</p></div

Number of patients susceptible to etravirine and rilpivirine.

<p>Number of patients susceptible to etravirine and rilpivirine.</p

Baseline characteristics of the patients.

<p>Baseline characteristics of the patients.</p

Top 10 NNRTI-RAMs found in patients who failed either the NVP- or EFV-based regimen.

<p>Top 10 NNRTI-RAMs found in patients who failed either the NVP- or EFV-based regimen.</p

Susceptibility pattern of etravirine and rilpivirine in patients who failed nevirapine and efavirenz-based regimens.

<p>Susceptibility pattern of etravirine and rilpivirine in patients who failed nevirapine and efavirenz-based regimens.</p

Overlaid Lateral Flow Immunoassay for the Simultaneous Detection of Two Variant-Specific SARS-CoV‑2 Neutralizing Antibodies

COVID-19 vaccines have been provided to the general public to build immunity since the 2019 coronavirus pandemic. Once vaccinated, SARS-CoV-2 neutralizing antibodies (NAbs-COVID-19) are needed for excellent protection against COVID-19. However, monitoring NAbs-COVID-19 is complicated and requires hospital visits. Moreover, the resulting NAbs-COVID-19 are effective against different strains of COVID-19 depending on the type of vaccine received. Here, an overlaid lateral flow immunoassay (O-LFIA) was developed for the simultaneous detection of two NAbs-COVID-19 against different virus strains, Delta and Omicron. The O-LFIA was visualized with two T-lines with a single device using competition between the free antigen and the antigen-binding antibody. Angiotensin-converting enzyme 2 (ACE2) immobilized on the T-line binds to the antigen remaining after antibody binding. Under the optimum conditions, the proposed device exhibited 50% inhibition concentrations (IC50 values) of 45.1 and 53.6 ng/mL for the Delta and Omicron variants, respectively. Additionally, the proposed platform was applied to real-world samples of animal and human serum, and the developed immunoassay provided results that were in good agreement with those obtained with the standard method. In conclusion, this developed O-LFIA can be used as an alternative method to detect NAbs-COVID-19 and can be enabled for future advancements toward commercialization