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An Introduction to Therapeutic Chimeric Antigen Receptor T Cells

GenScript Joins the Fight Against HIV

What is HIV?

The Human Immunodeficiency Virus (HIV) is a lentivirus which eventually progresses into the deadly disorder Acquired Immunodeficiency Syndrome (AIDS). HIV infects its host through exchange of bodily fluids commonly by sexual intercourse or childbirth. Because HIV is so easily transmittable, the virus continues to spread, currently infecting more then 37 million people world wide.

After HIV enters the body, it will infect specific cells of the immune system, such as CD4+ helper T-cells, macrophages, and dendritic cells. Upon infection, these immune cells will quickly lose their functionality and eventually die, leaving the patient with an extremely low level of active immune cells to fight entering infectious agents. Once a patients CD4+ count has reached a dangerously low level they will be diagnosed with AIDS and have to be continuously hospitalized to prevent future infections. Unfortunately, 70% of patients will succumb to an AIDS related infection and die within 10 years of HIV infection.

The Structure of HIV

As shown below in figure 1, an HIV viroid is composed of numerous layers. The inner most layer is composed of two single strands of viral RNA surrounded by reverse transcriptase and integrase. The viral RNA is encompassed by a viral capsid, which itself is enclosed by the viral matrix. On the outside of the matrix is the viral envelope, composed a lipid bilayer. Imbedded within the lipid membrane, are glycoproteins essential for host cell docking, fusion, and penetration, as well as left over proteins from the previous host cells membrane.

Figure 1: Structure of the HIV Virus.

The Life Cycle of HIV

HIV infection begins when viral envelope glycoproteins bind with host cell receptors and induce fusion of the virus’ envelope with the host cell’s membrane. After fusion, the HIV viroid will be endocytosed into the cell where its envelope proteins will be removed and digested, leaving only the capsid coated RNA and proteins. Within the cytoplasm, viral RNA will be reverse transcribed into DNA, which will be taken up by the host cell’s nucleus and integrated into its genome. Once the host cell DNA goes through a round of transcription, the resulting viral RNA will be released and encapsulated by the viral envelope proteins eventually generated from host cell translation of RNA specific genes. The resulting virion will bind to the host cell’s membrane in order to be released through an exocytosis like mechanism.

Life Cycle of HIV

Figure 2: Life Cycle of HIV

The Need for an HIV Vaccine

HIV is a lentivirus, a genus of retrovirus’,which survives through injection and translation of its own RNA to generate new viral particles. Therefore, it is commonly treated through a combination of antiretrovirals (ARVs). Unfortunately, most patients have to constantly optimize their specific ARV schedule to combat their specific strain of HIV. This is because HIV has a rapid mutation rate, which can lead to novel strains which can become drug resistant. In order to combat HIV infection, researchers believe that generating a preventative vaccine will be much more effective than new ARVs, however generating a vaccine which prevents over 200 different strains of one virus has been a cumbersome task.

The NIH’s Vaccine Research Centers’ Astonishing Discovery

The NIH’s Vaccine Research Center (VRC) focuses on discovery and generation of vaccines for devastating diseases, spending a majority of their efforts on finding a vaccine for HIV. However, rather than relying on traditional methods of vaccine discovery, the scientists at VRC decided to take a reverse screen approach. Through this method, VRC analyzed the structural components of broadly neutralizing antibodies (BNAs) naturally developed in patients living with long term HIV infections. Their analysis revealed that the epitope of many of the BNAs was centered around the viral envelope and various components viral entry machinery. Further analysis of these regions revealed that a majority of this area was hidden prior to viral docking, making it difficult to use as a vaccine target. However, researchers did identify an exposed 8 amino acid long chain at the N-terminal of the Viral Entry Machineries Fusion Peptide (FP), the region responsible for fusion between the viral envelope and the host cells membrane. This discovery prompted VRC scientists to reach out to GenScript to assist in generating immunogens specific to this region as well as to generate the perfect immunization protocol to generate a wide breadth of broadly neutralizing antibodies; essentially creating a vaccine against HIV-1.

HIV-1 Envelope Membrane Fusion

Figure 3. HIV-1 Envelope Membrane Fusion. Researchers at VRC were interested in using the 8 amino acid N-terminal of the viral entry machineries fusion peptide (FP) as a vaccine immunogen. This decision was based on the identification that other regions of the FP was hidden until the GP120 trimer (envelope glycoprotein) binded with CD4 present on the host cells membrane, as shown in the above figure. By using the pre-bound exposed FP region, the vaccine could potentially inhibit the virus from fusing and infecting future host cells.

GenScript’s Contributions to VRC’s HIV Vaccine Development

When VRC contacted GenScript back in 2016 they were looking for a provider to assist in multiple aspects of their HIV vaccine development platform. First, GenScript needed to assist in generating multiple forms of the 8 amino acid region of the HIV fusion peptide to be tested for immunogenicity and antigenicity. Specifically, GenScript generated the HIV-1 fusion peptides with either a His tag or a free amine at the N terminal to be used for conjugation and immunization. Once the three peptide containing immunogens were generated (Env-Trimer, epitope scaffold, and keyhole limpet hemocyanin conjugated FP) GenScript performed and optimized all subsequent immunizations. In order to ensure each immunization produced the largest amount of broadly neutralizing antibodies, GenScript enhanced numerous parts of the immunization methodology. First, they used their proprietary Germinal Center Associated Nuclear Protein (GANP) mice for all immunizations, to ensure the production of high affinity and high specificity antibodies. Next, they used their self developed adjuvant to enhance immunogenicity of all tested antigens.

After deciding to use GenScript’s GANP mice and GS-adjuvant, GenScript also assisted in finalizing the immunization schedule for the co-immunization of the epitope scaffold and the FP-KLH as well as immunization with FP-KLH alone. After GenScript’s production team performed these immunizations, they selected the highest responding animals to be used for subsequent hybridoma generation and antibody purification. The resulting antibodies were then used for neutralization analysis, protein crystal screening, genetic and structural analysis. It is this collaboration which allowed GenScript to be noted as authors in VRC’s 2018 paper, ""Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize.

Check Out Our Summary of VRC’s Manuscript in Nature!

Read the full paper: Xu, Kai, et al. "Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1." bioRxiv (2018): 306282.
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