Ultrasensitive quantitative HIV-1 p24 antigen assay adapted to dried plasma spots to improve treatment monitoring in low-resource settings.

BACKGROUND: Our group has previously developed a quantitative and ultrasensitive HIV-1 p24 antigen assay that is inexpensive, easy-to-perform, and can be carried out in low-resource settings. Since antiretroviral therapies are becoming more accessible in resource-constrained countries, methods to assess HIV-1 viraemia are urgently needed to achieve a high standard of care in HIV-1 management. OBJECTIVES: To adapt our quantitative assay to dried plasma spots (DPS), in order to further simplify this test and make it more accessible to resource-constrained countries. STUDY DESIGN: DPS from 47 HIV-seropositive, treated or untreated adult individuals and 30 healthy individuals were examined. RESULTS: A specificity of 100% was observed when p24 antigen was measured using DPS, and no differences of p24 concentration could be seen between DPS and venous plasma. The correlation between DPS and venous plasma p24 was excellent (R=0.93, CI(95%)=0.88-0.96, p<0.0001). Similarly, p24 antigen concentrations using DPS were well correlated with RNA viral load (R=0.53, CI(95%)=0.27-0.72, p=0.0002). CONCLUSIONS: This quantitative p24 antigen test has similar sensitivity and specificity using DPS and venous plasma, and has the potential to improve health care delivery to HIV-affected individuals in resource-constrained countries.

An immune competent mouse model for the characterization of recombinant measles vaccines.

Today, immune compromised interferon-α-receptor deficient mice expressing hCD46 (IFNARCD46tg) are usually used for measles virus (MV) based vaccine characterization. However, for the development of MV-based recombinant vaccine candidates (rMV), an immune competent mouse model is desirable in order to induce and evaluate meaningful immune response. In this study, humoral and cellular immune response induced by rMV in immune competent mice expressing human MV receptor CD46 (hCD46tg) were compared with those induced in wild-type black/6, and IFNARCD46tg mice.   All three strains developed humoral and cellular response against MV, whereas only hCD46tg and IFNARCD46tg mice developed a humoral response against the transgene. Differences were observed in the magnitude of the response, where the IFNARCD46tg mice displayed the strongest immune responses, followed by the hCD46tg mice and the black/6 mice. Interestingly, hCD46tg and wt black/6 mice showed a predominant CD4(+) T-cell response against MV-N, whereas IFNARCD46tg mice developed both, CD4(+) and CD8(+) T-cell response against MV-N. Analysis of the cytokine profile of MV-N specific CD4(+) T-cells and transgene (SIVgag) specific CD8(+) T-cells revealed qualitative differences of the T-cell responses; noticeably a significant reduction of the frequency of CD4(+)IL-2(+) expressing cells in IFNARCD46tg mice as compared with hCD46tg or wt black/6 mice. We show in this study significant quantitative and qualitative differences in immune responses between immune competent and immune-compromised mice. Our results therefore highlight the importance of the animal model and support the use of hCD46tg mice as mouse model for the characterization of the immunological profile induced by recombinant measles virus vaccine candidates.

Relevance of a pre-existing measles immunity prior immunization with a recombinant measles virus vector.

Measles virus (MV) vectors are promising candidates for designing new recombinant vaccines since the parental live vaccines have a well-known safety and efficacy record. Like all viral vectors, the MV vector efficacy in inducing a protecting immune answer could be affected by the pre-existing immunity among the human population. In order to determine the optimal immunization route and regimen, we mimicked a MV pre-immunity by passively administrating MV neutralizing antibodies (MV-nAb) prior intramuscular (i.m.) and/or intranasal (i.n.) immunization with recombinant MV expressing the SIV-gag antigen (rMV-SIVgag). Our results revealed that 500 mIU of MV-nAb allowed the induction of a humoral and cellular immune response against the vector and the transgene, while higher titers of the MV-nAb were significantly inhibitory. In a prime-boost regimen, in the presence of MV-nAb, the intranasal-intramuscular (i.n.-i.m.) or intramuscular-intramuscular (i.m.-i.m.) routes induced higher humoral immune responses against the vector and the transgene (SIV-gag). In naive animals, cellular immune response was significantly higher by i.m. immunization; however, MV pre-immunity did not seem to affect the cellular immune response after an i.n. immunization.   In summary, we show that a pre-existing immunity of up to 500 mIU anti-MV neutralizing antibodies had little effect on the replication of rMV and did not inhibit the induction of significant humoral and cellular immune responses in immune-competent mice.

Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector.

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 10 ( 20) . The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors.

Adaptation of the ultrasensitive HIV-1 p24 antigen assay to dried blood spot testing.

Implementation of molecular tests for the assessment of pediatric HIV-1 infection in resource-limited countries is difficult because of technical complexity and costs. Alternatives like the ultrasensitive HIV-1 p24 antigen enzyme-linked immunosorbent assay have therefore been proposed. We have now adapted this test to dried blood spot (DBS) plasma p24 antigen (p24). High background activity was recognized as originating from endogenous peroxidase and eliminated by H2O2 quenching. The assay was evaluated with 72 pediatric specimens from Tanzania and with 210 pediatric or adult specimens from Switzerland. A real-time polymerase chain reaction assay for DBS DNA and/or plasma RNA identified HIV-1 infection in 38 Tanzanian children. HIV-1 subtypes included 18 C, 9 A1, 8 D, 1 AC, 1 J-like, and 1 unidentified. The detection rates for the different assays were as follows: DBS-p24, 32 (84%) of 38 samples; DBS DNA, 30 (79%) of 38 samples; plasma-p24, 23 (85%) of 27 samples; and plasma RNA, 30 (100%) of 30 samples. False-negative DBS-p24 was associated with subtype D (P < 0.01). DBS-p24 detection for non-D subtypes was 93% (95% confidence interval: 81% to 99%), and for subtype C, it was 94% (95% confidence interval: 76% to 99%). Specificity among 193 HIV-negative DBS samples was 100%. Correlation of DBS-p24 and plasma-p24 concentrations was excellent (R = 0.83, P < 0.0001). DBS-p24 is thus a promising alternative to molecular tests for HIV-1 in subtype C regions. It should now be evaluated in large studies of children for accurate assessment of diagnostic sensitivity.

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo.

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.