Constituents of Croton megalocarpus with Potential Anti-HIV Activity.

Reported herein is an anti-HIV monochlorinated compound, 1ß-acetoxy-3ß-chloro-5α,6α-dihydroxycrotocascarin L (1), of the rare crotofolane diterpenoid class. Compound 1, a suspected artifact of extraction, along with the previously undescribed 11ß-acetoxycrotocascarin L (2) and a known compound, crotocascarin K (3), were isolated from the bark of Croton megalocarpus, a Kenyan oil-producing seed crop. Compounds 1 and 3 inhibited HIV-1 replication with IC50 values of 28 and 5.5 nM, respectively. Furthermore, both compounds lacked cytotoxicity toward MT-4 cells and FM-55-M1 cells at concentrations of up to 50 µM. Compounds 1 and 3 were both found to inhibit HIV-1 protease.

Anti-HIV Ermiasolides from Croton megalocarpus.

In recent years, elucidation of novel anti-HIV bioactive compounds from natural products is gaining importance rapidly, not only from the research and publications, but also from controlled clinical studies. Here we report three new anti-HIV eudesmane-type sesquiterpenes, 5ß-Hydroxy-8α-methoxy eudesm-7(11)-en-12,8-olide (1), 5ß,8α-Dihydroxy eudesm-7(11)-en-12,8-olide (2) and 5ß-Hydroxy-8H-ß-eudesm-7(11)-en-12,8-olide (3). These are trivially named ermiasolide A-C and were isolated from the bark of Croton megalocarpus. 5ß-Hydroxy-8α-methoxy eudesm-7(11)-en-12,8-olide (1), showed the highest anti-HIV activity by inhibiting 93% of the viral replication with an IC50 = 0.002 µg/mL. On the other hand, 5ß-Hydroxy-8H-ß-eudesm-7(11)-en-12,8-olide (3) and 5ß,8α-dihydroxy eudesm-7(11)-en-12,8-olide (2), inhibited viral replication by 77.5% at IC50 = 0.04 µg/mL and 69.5% at IC50 = 0.002 µg/mL, respectively. Molecular docking studies showed that the proposed mechanism of action leading to these results is through the inhibition of HIV-protease.

Hepatitis C virus genotypes in Kenya.

Hepatitis C virus is a great public-health concern worldwide. Phylogenetic analysis of the HCV genome has identified six different genotypes that have generally been divided into several subtypes. There is very little information on HCV seroprevalence and genotypes in Kenya. To determine the genotypes of HCV circulating in Kenya, blood donor samples were serologically tested and confirmed by polymerase chain reaction (PCR). Positive samples were cloned and sequenced, and phylogenetic analysis conducted to determine the HCV genotypes. One hundred Murex-seropositive samples were re-tested using a passive hemagglutination test, and 16 of these were identified as seropositive. Further testing of all of the samples by PCR identified only 10 of the 16 samples as positive. Thus, only 10 % (10/100) of the samples were viremic. Six were from females (60 %), and four were from males (40 %). The mean age of the positive donors was considerably low, at 25 +/- 9 years. Genotypic testing indicated the presence of genotype 1a (10 %) and genotype 2b (90 %). This study reports on HCV genotypes in a blood donor population in Kenya where little had been done to provide information on HCV genotypes.

Anti-HIV crotocascarin ω from Kenyan Croton dichogamus.

An anti-HIV methanol-soluble fraction of a 1:1 CH2Cl2:CH3OH extract of twigs of a Kenyan Croton dichogamus yielded seven compounds, the new crotocascarin ω (1), the known ß-oplopanone (2), dihydroconiferyl acetate (3), 3'(4''-hydroxyphenyl)-propyl benzoate (4), lupeol, sitosterol and stigmasterol. Crotocascarin ω (90%) inhibited HIV-1 replication with an IC50 value of 5.3 nM, and the compound was cytotoxic towards MT-4 cells presenting an IC50 value of 84 µM. In silico modelling showed that the anti-HIV activity for compound 1 could be through the HIV-1 protease inhibition.

Cytotoxicity and anti-HIV activities of extracts of the twigs of Croton dichogamus Pax.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) is a clinical syndrome resulting from infection with human immunodeficiency virus (HIV), which causes profound immunosuppression. Anti-HIV drugs that are currently available are chemically synthesized and are frequently limited by side effects, the emergence of drug resistance, affordability, and availability, with over 5 million people in the world lacking access to treatment. As a result, to discover new anti-HIV agents, we investigated the effects of Kenyan C. dichogamus extracts on the laboratory-adapted strain HIV-1IIIB in human T-lymphocytic MT-4 cells. METHODS: Four soluble fractions of 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus Pax were tested for their replication inhibition activity against the laboratory-adapted strain HIV-1IIIB in the human T-lymphocytic MT-4 cell line. The plant extracts were further evaluated for their cytotoxicity in MT-4 cells using the MTT assay. RESULTS: The cytotoxicity CC50 values of the methanol and methylene chloride soluble fractions of C. dichogamus were found to be between 19.58 ± 0.79 and 167 ± 0.8 µg/ml, respectively. The hexane, methylene chloride, and methanol soluble fractions of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus showed inhibition of the HIV-1IIIB laboratory-adapted strain in a virus-infected cell culture antiviral assay. The methanol soluble fraction of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus showed significant anti-HIV activity by inhibiting more than 90% of viral-induced cytopathic effects with an IC50 value of 0.06 ± 0.01 µg/ml, giving an SI of 318.5. CONCLUSION: Based on our findings, the methanol soluble fraction of the 1:1 v/v CH2Cl2:MeOH extract of the twigs of C. dichogamus has shown potential efficacy in inhibiting viral replication and could be considered a promising candidate for further studies.

In vitro anti-HIV and cytotoxic effects of pure compounds isolated from Croton macrostachyus Hochst. Ex Delile.

Croton macrostachyus is an important plant in traditional African medicine, widely utilized to treat a variety of diseases. In Kenya, HIV-infected patients use leaf and root decoctions of the plant as a cure for cough, back pain, bleeding, skin diseases, warts, pneumonia, and wounds. This study aimed to evaluate the anti-HIV activities and cytotoxic effects of extracts and chemical constituents isolated from C. macrostachyus. In our previous study we demonstrated that the hexane, CH2Cl2, ethyl acetate and methanol soluble fractions of a 1:1 v/v/ CH2Cl2/MeOH crude extracts of the leaves and stem bark of C. macrostachyus exhibited potent anti-HIV activities against HIV-1 with IC50 values ranging from 0.02-8.1 µg/mL and cytotoxicity effects against MT-4 cells ranging from IC50 = 0.58-174 µg/mL. Hence, hexane soluble extract of 1:1 v/v/ CH2Cl2/MeOH crude extract of the leaves of C. macrostachyus, that was more potent against HIV-1 at IC50 = 0.02 µg/mL was subjected to column chromatography leading to the isolation of 2-methoxy benzyl benzoate (1), lupenone (2), lupeol acetate (3), betulin (4), lupeol (5), sitosterol (6) and stigmasterol (7). Lupenone (2), lupeol acetate (3) and betulin (4) exhibited anti-HIV-1 inhibition at IC50 = 4.7 nM, 4.3 and 4.5 µg/mL respectively. The results obtained from this study support the potential of C. macrostachyus, as a source of anti-HIV constituents.

In Vitro Cytotoxicity and Anti-HIV Activity of Crude Extracts of Croton macrostachyus, Croton megalocarpus and Croton dichogamus.

INTRODUCTION: Human immunodeficiency virus (HIV) affects the body's defense mechanisms and leads to a number of opportunistic infections which later cause fatality as a result of an acquired immunodeficiency syndrome (AIDS). More than half a million individuals have lost their life in 2020 due to this disease. Antiretroviral drugs have played a great role in improving the quality of life of HIV infected individuals. The side effects of these drugs coupled with resistance of the virus to the various regimens, necessitates the search for potentially new and effective antiretroviral medication. The objective of this study is to evaluate anti-HIV activity of crude extracts of three Croton plants. METHODS: As part of our effort in screening anti-HIV medications, we evaluated the cytotoxicity and anti-HIV activity of three Croton species used as herbal medicine in Africa. Crude extracts of Croton macrostachyus, Croton megalocarpus and Croton dichogamus were tested for their replication inhibition activity against laboratory adapted strains HIV-1IIIB in Human T-lymphocytic MT-4 cell line. RESULTS: Based on our findings, the crude aerial part extract of C. dichogamus displayed the highest anti-HIV activity by inhibiting 73.74% of viral induced cytopathic effect (CPE) at IC50 value of 0.001 + 0.00 µg/mL giving a selectivity index (SI) of 3116.0. In addition, the crude leaf extract of C. megalocarpus showed higher anti-HIV activity by inhibiting 74.65% of CPE at IC50 value of 0.05 + 0.03 µg/mL giving an SI of 571.3. CONCLUSION: Out of five extracts from three Croton species screened for anti-HIV activity using human T-lymphocytic MT-4 cells, the leaf extract of Croton megalocarpus and aerial part extract of Croton dichogamus could be considered as promising extracts as they display high antiviral activity with low toxicity and high selectivity index values. To investigate the active constituents responsible for the anti-HIV activity, chemical identification of the active constituents is now in progress in our laboratory. Since there is no previously reported anti-HIV activity for these plants, there is a great need to isolate the compounds responsible for the noted activity.

A Review of Current Strategies Towards the Elimination of Latent HIV-1 and Subsequent HIV-1 Cure.

BACKGROUND: During the past 35 years, highly effective ART has saved the lives of millions of people worldwide by suppressing viruses to undetectable levels. However, this does not translate to the absence of viruses in the body as HIV persists in latent reservoirs. Indeed, rebounded HIV has been recently observed in the Mississippi and California infants previously thought to have been cured. Hence, much remains to be learned about HIV latency, and the search for the best strategy to eliminate the reservoir is the direction current research is taking. A systems-level approach that fully recapitulates the dynamics and complexity of HIV-1 latency In vivo and is applicable in human therapy is prudent for HIV eradication to be more feasible. OBJECTIVES: The main barriers preventing the cure of HIV with antiretroviral therapy have been identified, progress has been made in the understanding of the therapeutic targets to which potentially eradicating drugs could be directed, integrative strategies have been proposed, and clinical trials with various alternatives are underway. The aim of this review is to provide an update on the main advances in HIV eradication, with particular emphasis on the obstacles and the different strategies proposed. The core challenges of each strategy are highlighted and the most promising strategy and new research avenues in HIV eradication strategies are proposed. METHODS: A systematic literature search of all English-language articles published between 2015 and 2019, was conducted using MEDLINE (PubMed) and Google scholar. Where available, medical subject headings (MeSH) were used as search terms and included: HIV, HIV latency, HIV reservoir, latency reactivation, and HIV cure. Additional search terms consisted of suppression, persistence, establishment, generation, and formation. A total of 250 articles were found using the above search terms. Out of these, 89 relevant articles related to HIV-1 latency establishment and eradication strategies were collected and reviewed, with no limitation of study design. Additional studies (commonly referenced and/or older and more recent articles of significance) were selected from bibliographies and references listed in the primary resources. RESULTS: In general, when exploring the literature, there are four main strategies heavily researched that provide promising strategies to the elimination of latent HIV: Haematopoietic Stem-Cell Transplantation, Shock and Kill Strategy, Gene-specific transcriptional activation using RNA-guided CRISPR-Cas9 system, and Block and Lock strategy. Most of the studies of these strategies are applicable in vitro, leaving many questions about the extent to which, or if any, these strategies are applicable to complex picture In vivo. However, the success of these strategies at least shows, in part, that HIV-1 can be cured, though some strategies are too invasive and expensive to become a standard of care for all HIV-infected patients. CONCLUSION: Recent advances hold promise for the ultimate cure of HIV infection. A systems-level approach that fully recapitulates the dynamics and complexity of HIV-1 latency In vivo and applicable in human therapy is prudent for HIV eradication to be more feasible. Future studies aimed at achieving a prolonged HIV remission state are more likely to be successful if they focus on a combination strategy, including the block and kill, and stem cell approaches. These strategies propose a functional cure with minimal toxicity for patients. It is believed that the cure of HIV infection will be attained in the short term if a strategy based on purging the reservoirs is complemented with an aggressive HAART strategy.

HIV-1 subtype and viral tropism determination for evaluating antiretroviral therapy options: an analysis of archived Kenyan blood samples.

BACKGROUND: Infection with HIV-1 is characterized by genetic diversity such that specific viral subtypes are predominant in specific geographical areas. The genetic variation in HIV-1 pol and env genes is responsible for rapid development of resistance to current drugs. This variation has influenced disease progression among the infected and necessitated the search for alternative drugs with novel targets. Though successfully used in developed countries, these novel drugs are still limited in resource-poor countries. The aim of this study was to determine HIV-1 subtypes, recombination, dual infections and viral tropism of HIV-1 among Kenyan patients prior to widespread use of antiretroviral drugs. METHODS: Remnant blood samples from consenting sexually transmitted infection (STI) patients in Nairobi were collected between February and May 2001 and stored. Polymerase chain reaction and cloning of portions of HIV-1 gag, pol and env genes was carried out followed by automated DNA sequencing. RESULTS: Twenty HIV-1 positive samples (from 11 females and 9 males) were analyzed. The average age of males (32.5 years) and females (26.5 years) was significantly different (p value < 0.0001). Phylogenetic analysis revealed that 90% (18/20) were concordant HIV-1 subtypes: 12 were subtype A1; 2, A2; 3, D and 1, C. Two samples (10%) were discordant showing different subtypes in the three regions. Of 19 samples checked for co-receptor usage, 14 (73.7%) were chemokine co-receptor 5 (CCR5) variants while three (15.8%) were CXCR4 variants. Two had dual/mixed co-receptor use with X4 variants being minor population. CONCLUSION: HIV-1 subtype A accounted for majority of the infections. Though perceived to be a high risk population, the prevalence of recombination in this sample was low with no dual infections detected. Genotypic co-receptor analysis showed that most patients harbored viruses that are predicted to use CCR5.

Molecular genetic diversity of hepatitis B virus in Kenya.

Eight genotypes of hepatitis B virus (A-H) and subgenotypes have been recognized worldwide. However, there is limited information on prevalent genotypes in many countries in Africa. This study was undertaken to determine the hepatitis B virus (HBV) genotypes in Kenya. Seropositive HBV blood samples from a blood donor setting were used in the study. HBV genotypes were determined in 52 nucleic acid-positive samples using specific primer in a nested PCR and sequencing employed in the HBV genotyping. This study shows presence of HBV variants with genotypes A (88%), E (8%) and D (4%). In conclusion, we found that HBV genotype A is the most predominant genotype in Kenya with both subgenotype A1 and A2 present. Genotype D and E are also present in our population. This demonstrates that there could be a high genetic diversity of HBV in Kenya.

An in vitro evaluation of drugs used in the Kenyan ART program.

The majority of anti-HIV drug susceptibility tests have been performed on subtype B HIV-1 strains, since these are the most prevalent in countries designing, testing, and manufacturing the current anti-HIV agents. The increasing global spread of HIV subtype highlights the need to determine the activity of anti-HIV drugs against subtypes of HIV other than subtype B. Furthermore an increasing number of individuals infected with many of the non subtype B virus strains now receive antiretroviral therapy because of rollout programs in developing countries as well as increasing migration to the developed world. The phenotypic susceptibility of two laboratory strains HIV-1JFRL and HIV-1IIIB (representing subtype B) and two clinical isolates HIV-104RTA and HIV-1025RTA (representing subtypes A and D respectively) was determined. The in vitro drug susceptibility testing of the isolates was carried out in C8166 cell line and in peripheral blood mononuclear cells (PBMCs). The study revealed that the drugs used in the Kenyan national ART program inhibited HIV-1 replication in-vitro as their inhibitory concentrations (IC50) compared well with the standard Inhibitory concentration values. The results also suggest a biochemical similarity of the reverse transcriptase (RT) and protease enzymes from these subtypes despite the divergence at the genetic level. The findings suggest that similar clinical benefits of antiviral therapy obtain in persons infected with other subtypes of HIV-1other than subtype B and that the generic drugs used in the national ART program in Kenya are as efficacious as branded drugs in inhibiting HIV replication in vitro despite the limited number of the viruses studied.

HIV type 1 subtypes in circulation in northern Kenya.

The genetic subtypes of HIV-1 circulating in northern Kenya have not been characterized. Here we report the partial sequencing and analysis of samples collected in the years 2003 and 2004 from 72 HIV-1-positive patients in northern Kenya, which borders Ethiopia, Somalia, and Sudan. From the analysis of partial env sequences, it was determined that 50% were subtype A, 39% subtype C, and 11% subtype D. This shows that in the northern border region of Kenya subtypes A and C are the dominant HIV-1 subtypes in circulation. Ethiopia is dominated mainly by HIV-1 subtype C, which incidentally is the dominant subtype in the town of Moyale, which borders Ethiopia. These results show that cross-border movements play an important role in the circulation of subtypes in Northern Kenya.

Isolation and biological characterization of non-B HIV type 1 from Kenya.

The isolation and characterization of primary strains of human immunodeficiency virus (HIV) is a vital tool for assessing properties of viruses replicating in HIV-infected subjects. HIV-1 isolation was carried out from 30 HIV-1-infected patients from a Comprehensive Care Clinic (CCC) after informed consent. Virus was successfully isolated from 9 out of the 30 samples investigated. Seven of the isolates were from drug-naive patients while two were from patients on antiretroviral drugs. The isolates were biologically phenotyped through measurement of the syncytium-inducing capacity in MT2 cells. Six of the isolates exhibited syncytia induction (SI) associated with CXCR4 coreceptor usage while three of the isolates were non-syncytia-inducing (NSI) isolates associated with CCR5 coreceptor usage. In addition, the replication capacity of the isolates was further determined in established cell line CD4(+) C8166. Indirect immunofluorescence assay was used to check the antigen expression on the cells as a supplementary test. HIV-1 isolation success was 70% (7/10) and 20% (2/20) in naive and drug-experienced patients, respectively. The majority of the viral isolates obtained (6/9) were of the SI phenotype, though SI virus strains are rare among non-B subtypes. A significant correlation between virus isolation success and viral load was established. Coreceptor use data for heavily treatment-experienced patients with limited treatment options are scanty and this is the group with perhaps the most urgent need of novel antiretroviral agents.

Switch from 200 to 350 CD4 baseline count: what it means to HIV care and treatment programs in Kenya.

INTRODUCTION: With the increasing population of infected individuals in Africa and constrained resources for care and treatment, antiretroviral management continues to be an important public health challenge. Since the announcement of World Health Organization recommendation and guidelines for initiation of antiretroviral Treatment at CD4 count below 350, many developing countries are adopting this strategy in their country specific guidelines to care and treatment of HIV and AIDS. Despite the benefits to these recommendations, what does this switch from 200 to 350 CD4 count mean in antiretroviral treatment demand? METHODS: A Multi-centre study involving 1376 patients in health care settings in Kenya. CD4 count was carried out by flow cytometry among the HIV infected individuals in Kenya and results analyzed in view of the In-country and the new CD4 recommendation for initiation of antiretroviral treatment. RESULTS: Across sites, 32% of the individual required antiretroviral at <200 CD4 Baseline, 40% at <250 baseline count and 58% based on the new criteria of <350 CD4 Count. There were more female (68%) than Male (32%).Different from <200 and <250 CD4 baseline criteria, over 50% of all age groups required antiretroviral at 350 CD4 baseline. Age groups between 41-62 led in demand for ART. CONCLUSION: With the new guidelines, demand for ARVs has more than doubled with variations noted within regions and age groups. As A result, HIV Care and Treatment Programs should prepare for this expansion for the benefits to be realized.

HIV type 1 gag genetic diversity among antenatal clinic attendees in North Rift Valley, Kenya.

HIV genetic recombination and high mutation rate increase diversity allowing it to escape from host immune response or antiretroviral drugs. This diversity has enabled specific viral subtypes to be predominant in specific regions. To determine HIV-1 subtypes among seropositive antenatal clinic attendees in Kenya's North Rift Valley, a cross-sectional study was carried out on 116 HIV-1-positive blood samples. Proviral DNA was extracted from peripheral blood mononuclear cells by DNAzol lysis and ethanol precipitation. Polymerase chain reactions using specific primers for HIV-1 gag and population sequencing on resulting amplicons were carried out. Phylogenetic analysis revealed that 81 (70%) were subtype A1, 13 (11%) subtype D, 8 (7%) subtype C, 3 (3%) subtype A2, 1 (1%) subtype G, and 10 showed possible recombinants: 5 (4%) subtype A1D, 4 (3%) subtype A1C, and 1 (1%) subtype A2C. These data support the need to establish circulating subtypes for better evaluation of effective HIV diagnostic and treatment options in Kenya.

The changing trend of HIV type 1 subtypes in Nairobi.

Monitoring the distribution of HIV-1 subtypes and recombinants among infected individuals has become a priority in HIV therapy. A laboratory analysis of samples collected from HIV-positive patients attending an STI clinic in Nairobi was done between March and May 2004. PCR was carried out on pol (intergrase) and env (C2V3) regions and resulting data on the 54 samples successfully analyzed revealed the following as circulating subtypes: 35/54(65%) were A1/A1, 5/54(9%) were A/C, 4/54 (7%) were A1/D, 1/54 (2%) was C/D, 1/54 (2%) was D/D, 1/54 (2%) was A1/A2, 1/54 (2%)was G/G, 1/54 (2%) was A2/D, 1/54 (2%) was C/C, and 4/54 (7%) were CRF02_ AG. The results show an increase in HIV-1 recombinants with the emergence of A1/A2 and an increase in CRF02_AG recombinants. Subtype diversity in the advent of ARV use will impact negatively on treatment outcomes. As such, increased viral evolution and recombination will call for continuous evaluation of available anti-HIV regimens for better management of those infected with HIV-1.

Genetic diversity of HIV type 1 along the coastal strip of Kenya.

A study on the genetic diversity of HIV-1 subtypes present along the coastal strip of Kenya, i.e., Kilifi, Mombasa, Msambweni, and Malindi districts, was carried out. DNA sequences for regions encoding a portion of the env-gp41 region of the virus were generated by PCR and sequenced directly. Eighty six samples that were successfully sequenced were analyzed. From the analysis, 86% (74) were subtype A1, 5% (4) were subtype C, 8% (7) were subtype D, and 1% (1) was subtype G. This study shows that HIV-1 subtype A1 is the most dominant subtype in circulation in this region.