Difference between revisions of "Team:Montpellier/Antisperm/antibodies"

Line 137: Line 137:
 
                 </tr>
 
                 </tr>
 
               </table>
 
               </table>
             </div>
+
             <p>We chose 4 differents SP to perform the secretion of scFv :
           </div>
+
 
 +
            <ul>
 +
              <li>CbsA</li>
 +
              <p>The first that we chose is CbsA. This SP is used for secretion of antibodies in <i>Lactobacillus jensenii</i> (Ref : Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus ; Engineered Vaginal <i>Lactobacillus</i> Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N).<br/>
 +
              The SP is synthesized on the N-terminal domain. Downstream the CbsA sequence are added 4 amino acids (APVT) at the N-terminal domain of the scFv. These 4 amino acids are similar to a native signal peptidase cleavage site of this protein. The addition of the APVT sequence shown to improve the secretion of a full-length protein in <i>L. jensenii</i>.</p>
 +
 
 +
              <li>Epr, YncM, YjfA</li>
 +
              <p>These SPs are coming from a screening of SPs of <i>Bacillus subtilis</i>. As they can be used to secrete proteins in B. subtilis which is a gram positive bacteria, we decided to try to use them to secrete our stuff in L. jensenii that is also a gram positive bacteria. We used the same template for the construction. These SPs are on the N-terminal domain, and followed by the APVT sequence on their N-terminal domain.</p>
 +
            </ul>
 +
           </p>
 +
        </div>
 +
      </div>
  
 
       <h2>Experiments</h2>
 
       <h2>Experiments</h2>

Revision as of 12:57, 8 September 2018

Antibodies

Antibodies


Origin of antisperm antibodies


During the 20th century, several cases of infertile men and women were observed. At this time, the reasons of this sterility were elusive. Researchers discovered that this was the result of the production of antisperm antibodies (ASA). These antibodies can be produced in the body of women and men against spermatozoa, and they cause infertility. Presence of ASA in male and/or female partner have been considered as infertility cause in around 2-30% of infertile couples [1]. For example, ASA production in women can be caused after spermatozoal deposition into genital tract with a compromised epithelial barrier, the peritoneal cavity or the gastrointestinal tract.

Researchers tried to use these antibodies and related antigens to create a contraceptive vaccine (CV) [2]. They made mice produce ASA (production by B cells) by vaccinating them with the related antigen. The results show that vaccination with a sperm antigen or its cDNA causes a long-term, reversible contraception in female mice [3].

They showed that ASA can be very effective for a long-term and reversible contraception. This is exactly what we want to create by engineering L. jensenii to make them produce ASA against a spermatozoa antigen.

Why do we chose to synthesize ASA ?


What is and antibody ?

The classical representation of an antibody is as a Y-shaped molecule composed of four polypeptide subunits with two identical heavy and light chains, linked by disulfure bounds. Each chain is composed of a constant (constant heavy CH, constant light CL) and a variable part (variable heavy VH, variable light VL). It is secreted by B lymphocytes that specifically recognize and neutralize antigens.

scFv

scFv (for Single-Chain Variable Fragment) are recombinant molecules where the variable region of the heavy (VH) and light (VL) immunoglobulin chains are linked into a single peptide. VH and VL are linked with a flexible linker sequence, here Gly4Ser. Glycine amino-acids ensure a good flexibility of the linker. Effect of scFv is similar to antibodies.

Antisperm antibodies

ASA are immunoglobulins (IgG, IgA, IgM) directed against sperm antigens. The production can be caused whenever sperm encounter the immune system. They can have different effects.

Effect of ASA on sperm

ASA can affect fertility through several mechanisms : inhibition of sperm motility, capacitation, acrosome reaction, sperm zona interaction, sperm-oolemma binding and penetration and preimplantation embryonic development.
The main activity of spermicides is the inhibition of sperm motility. We chose several ASA that have this effect. If spermatozoa have their motility inhibited, they can not fertilize the egg anymore [2].

YLP20 and YLP12


First, we read publications to find several ASA. We found 4 interesting ASA with their related antigens.

We decided to choose YLP20 for a candidate as it is very well documented. Moreover YLP12 the antigen targeted by YLP20 is small so we can easily have this peptide synthesize to test the activity of the scFv that we want to produce.


Table : ASA and related antigens.
Antibody Related antigen
YLP20 YLP12
AFA-1 FA-1
FAB-7 FA-1
AS16 Human Sperm Extract (HSE)

YLP20

The YLP20 scFv that we want to produce is composed of a heavy chain (VH), a light chain (VL), a linker (Gly4Ser) and an E-Tag.

Role of each part :

  • E-Tag : purification of our scFv using anti-E-Tag antibody
  • linker Gly4Ser : link VH and VL
  • VH and VH : compose the antigen-binding domain

YLP12

Objectives


Production

We first want to engineer L. jensenii and B. subtilis to make them produce YLP20 scFv.

Secretion

The next step is to make L. jensenii and B. subtilis secrete YLP20 scFv in the supernatant using a signal peptide.

Signal Peptide Origin Amino acid sequences
CbsA L. jensenii MKKNLRIVSAAAAALLAVAPVAASAVSTVSA
Epr B. subtilis MKNMSCKLVVSVTLFFSFLTIGPLAHA
YncM B. subtilis MAKPLSKGGILVKKVLIAGAVGT
AVLFGTLSSGIPGLPAADA
YjfA B. subtilis MKRLFMKASLVLFAVVFVFAVKGAPAKA

We chose 4 differents SP to perform the secretion of scFv :

  • CbsA
  • The first that we chose is CbsA. This SP is used for secretion of antibodies in Lactobacillus jensenii (Ref : Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus ; Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N).
    The SP is synthesized on the N-terminal domain. Downstream the CbsA sequence are added 4 amino acids (APVT) at the N-terminal domain of the scFv. These 4 amino acids are similar to a native signal peptidase cleavage site of this protein. The addition of the APVT sequence shown to improve the secretion of a full-length protein in L. jensenii.

  • Epr, YncM, YjfA
  • These SPs are coming from a screening of SPs of Bacillus subtilis. As they can be used to secrete proteins in B. subtilis which is a gram positive bacteria, we decided to try to use them to secrete our stuff in L. jensenii that is also a gram positive bacteria. We used the same template for the construction. These SPs are on the N-terminal domain, and followed by the APVT sequence on their N-terminal domain.

Experiments


References
[1] Rajesh K. Naz, Subhash C.Chauhan. 2001. Presence of antibodies to sperm YLP12 synthetic peptide in sera and seminal plasma of immunoinfertile men. Molecular Human Reproduction Vol.7 no.1 pp. 21–26.
[2] Rajesh K. Naz. 2014. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide. Protein Science 2014 Vol.23:857—868.
[3] A.S. Samuel and R.K. Naz. 2008. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development. Human Reproduction Vol.23, No.6 pp. 1324–1337.
[4] Angela Marcobal, Xiaowen Liu, Wenlei Zhang, Antony S. Dimitrov, Letong Jia, Peter P. Lee, Timothy R. Fouts, Thomas P. Parks, and Laurel A. Lagenaur. 2016. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus. Aids Resaerch And Human Retroviruses Volume 32, Number 10/11.