Cyanovirin-N As A Solution For Suppression Of HIV Virus
Amongst numerous problems, the terrible AIDS pandemic that has continued to devastate the country remains the most disturbing to me as an individual. I recently got to learn that Kenya has the joint fourth largest HIV epidemic in the world with 1. 5 million people living with HIV as per the UNAIDS data of 2018. The number of AIDS related deaths remains notably high and the rate at which new infections are emerging especially amongst the youth is very alarming. A lot of effort has been channeled towards managing this disease and I strongly believe that recent advances in biotechnology and genomics have the ability to mitigate this long standing problem. The specific approach that can serve as a starting point is searching for new bioactive products that have antiviral activity in microorganisms present in nature. The pharmacodynamic properties of the new product(s) can be assessed and modified to suit therapeutic needs.
Having identified that the type of product needed is one with antiviral activity, the subsequent step would be to identify the best producing organism to investigate. With the knowledge of biological diversity in terms of species richness and ecosystem function, shotgun metagenomics can be very helpful to get largely unbiased samples of all genes from all members of a sampled community. With the advancement in techniques of DNA sequencing and the fall in prices of sequencing, investigation of the microbial ecology regarding the genes present can be done to a much higher scale and detail. Given that the data obtained from such metagenomics experiments is enormous, curation and extracting useful biological information through data mining will rely heavily on bioinformatics databases. The sample communities of microbes to be investigated should be those that are novel and rather neglected. Isolates from less explored ecosystems should be highlighted and the bioactive targets with antiviral activity should be matched with members of previously unscreened but known taxa, an example being cyanobacteria. Screening assays can then be done on the cyanobacteria.
Cyanovirin-N (CN-V) is one such protein with virus inactivating capability and is a gene product from cyanobacterium Nostoc ellipsosporum. This protein is able to block the interaction between HIV Virus type 1 with CD4 cells. It also has blocking action at the level of HIV-1 gp120 interaction with coreceptor (Chemokine Receptor type 5-CCR5) found on the surface of white blood cells. This is through inducing coreceptor down modulation after the CD4 binding step. The general mode of action of cyanovirin-N is blocking the membrane fusion reaction associated with HIV 1 entry. It is also nontoxic at concentrations much higher than those required for anti HIV activity which is a very attractive positive attribute. Cyanovirin-N has broad spectrum antiviral activity and is therefore not only specific to HIV. This only means that it has implications for potential clinical utility. Knowing all these properties of the protein the next challenge would be the most effective way to introduce it to the body of an infected patient such that it will have the ability to cause efficient viral suppression. Escherichia coli has been widely used as a recombinant protein expression system and the recombinant CN-V protein is indistinguishable from that produced by Nostoc ellipsosporum. Having an expression system like this one makes it possible to yield large scale production of clinical grade CV-N for research and as an anti-HIV microbicide. Different bacteria can also be bioengineered to secrete CN-V.
Lactic acid bacteria which are commensal organisms in the human gut and in the vaginal microbiome can therefore be genetically engineered to secrete the CN-V protein. Lactic acid bacteria are usually used as starter cultures in manufacture of yoghurt, therefore, the genetically engineered strains that have the ability to produce CN-V can be formulated as yoghurt. Standard fermentation procedures used to create yoghurt will still be maintained, the only difference being that Lactic acid bacteria with CN-V will comprise the starter culture. The yoghurt fermentation process must be done in such a way that the biosynthetic capacity of the genetically engineered bacteria is preserved. The technical process involved to make this possible starts by identifying the species of lactic acid bacteria that are commensal organisms in the human gut. These include Lactobacillus plantarum in the rectum and Lactobalillus jensenii and Lactobacillus gasseri in the vaginal microbiome. For efficient expression of the CV-N, I would start by first identifying the gene that produces this protein in Nostoc ellipsosporum. I would then isolate the mRNA for this gene, make a single copy of the complementary DNA using the reverse transcriptase enzyme then make another copy by adding the DNA polymerase enzyme. Alternatively, I can also isolate this specific gene from the genome of the cyanobacteria through fragmenting the DNA using restriction enzymes then identifying the fragment containing the desired gene. I would then insert a codon optimized CV-N gene into a plasmid with lactic acid bacteria specific signals which will enable production of the recombinant protein in large volumes. The properties of the plasmid that I would be keen to look at would be the origin of replication, presence of recognition sites for restriction enzymes for insertion of the DNA fragment, ability to replicate autonomously in the host and presence of markers for selection. I would then make the specific strains of lactic acid bacteria that I had previously identified and cultured electro competent. This can be done by treating the cells with calcium chloride at high temperatures. I would then screen these host cells to check for successful integration and replication of the CN-V gene and expression of the CN-V protein. Given that the plasmid vector chosen carries genes for antibiotics resistance, I would use antibiotics to select for presence of vector molecules. T
his entire process would result in a good number of genetically engineered lactobacillus which can be used as starter cultures in yoghurt production. This approach of mucosal delivery of the anti HIV 1 protein compound overcomes hurdles of commercial and clinical development of protein microbicides like a relatively short half-life of the microbicide upon direct mucosal application. Proteases in the body can easily destroy the protein rendering it ineffective. The delivery of this medication to the rectum upon oral intake of the bioengineered yoghurt can reduce HIV 1 transmission especially in amongst gay people. To ensure absolute efficiency of this approach, numerous antiviral molecules can be introduced in one specific lactic acid bacteria or a combination of numerous genetically engineered lactic acid bacteria each capable of secreting the antiviral protein can be introduced at once.
For a long time, the Kenya has embraced use of antiretroviral therapy and this has remained the go to solution for suppression of HIV virus. However, these antiretrovirals have quite a number of limitations, necessitating a more innovative approach to limit AIDS related deaths. These limitations include short and long term side effects which may even limit treatment. A number of life threatening side effects include elevated liver enzymes, extreme gastrointestinal intolerance, adverse metabolic effects for example insulin resistance and increased bleeding in hemophiliacs amongst others. An individual may also develop antiretroviral resistance due to high diversity of the HIV virus. Drug resistant HIV viruses are already present in the population since mutations occur spontaneously in the HIV genome. Antiretrovirals also demand very strict adherence as this is a major determinant of degree and duration of viral suppression. Most people infected with HIV are usually in denial for long and are affected by stigma hence suboptimal adherence is very common. The cost of the antiretroviral drugs is also quite high meaning those who may be willing to start taking the drugs might not have enough money to do so.
