Download Application of Biophysical techniques in AIDS Vaccine research:

Application of Biophysical techniques in AIDS Vaccine research:
Amit Anand
MOAC, University of Warwick
[email protected]
19th January 2005
ABSTRACT
At the moment the number of people dying because of human immuno-deficiency virus (HIV) in
a year is more than those dying due to natural calamities like floods, earth quake or volcano eruption.
Virology researchers are not only collaborating internationally but they are also joining hands with other
disciplines like physical sciences to solve the problem. Although the initial immune reaction against any
new deadly virus will be insignificant but it will definitely be present. Some rare but potent antibodies of
human origin which can neutralize HIV are been identified and their interactions are been visualized at
atomic level. Examples are b12, 2G12, 2F5, 4E10 and Z13
(9).
With the combination of
multidisciplinary approach, biophysical techniques are opening new horizons to explore every
possibility to solve the biggest challenge to the survival of human race on earth. This article discusses
some of the successes researchers have achieved with the help of biophysical techniques.
KEYWORDS: HIV Biophysical technique, X-ray crystallography, NMR, Mass Spectrometry
1
INTRODUCTION:
The success of the polio vaccine is attributed to the work of a great scientist Jonas Salk (19141995). Salk launched the first polio vaccine in 1952 which was followed by oral live polio vaccine by
Sabin in 1962 (1). In those days it was not possible to develop a vaccine strategically. Today by studying
the structure of HIV-1 along with its neutralizing monoclonal antibodies, scientist are trying to study the
possibility of making a successful vaccine.
IMMUNE MECHANISM:
Bacteria, virus, or any substance which is recognized as foreign invader by the body’s defense
mechanism has an immune reaction against it. Foreign substances are called antigens and as a defense,
body produces protein molecules called antibodies (immunoglobulin). These antibodies binds to specific
sites of the antigen and thus makes the antigen non reactive or neutralized
(2).
These specific sites on
antigens are called antigenic determinants or epitopes (3).
GENERAL STRUCTURE AND CLASSIFICATION OF IMMUNOGLOBULIN:
As is shown in the figure 1 below the basic structure of the antibody contains two identical light
chains and two identical heavy chains linked together by disulfide bonds, making a shape of Y. Based on
the structure of heavy chains immunoglobulin can be divided into five different classes: IgG, IgM, IgA,
IgD, and IgE.
2
Figure 1(a) The general structure of an antibody and (b) different immunoglobulins.
The figure is taken from Carl Branden, John Tooze-Introduction to protein structuresecond edition (4).
Figure 2 Schematic diagram of the production of antibodies.
The figure is taken from Carl Branden, John ToozeIntroduction to protein structure- second edition (4)
3
As shown in figure 2 antibodies are produce by B cells inside the human body. The important thing to
note is that each different antibody is produced only by a single clone of B cells (2) (5).
PRODUTION OF MONOCLONAL ANTIBODIES:
By somatic-cell hybridization of B-cells and T-cells, hybridomas were produced (B and T cells
are different type of immune cells). These hybridomas have some of the normal genes of B or T cells
and immortal-growth properties of cancer cells. Here the function of B cells is to produce monoclonal
antibodies and T cells secrete various growth factors (5) (6).
Figure 3 normal and cancerous hybridoma
cells. The figure is taken from
http://www.whfreeman.com/immunology/CH02/hy
bridomas.htm (6)
AIDS (ACQUIRED IMMUNODEFICIENCY SYNDROME):
AIDS is cause by the destruction of CD4+ lymphocytes by the human immunodeficiency viruses
(HIV-1, HIV-2, and the related simian immunodeficiency viruses SIV) in their respective host.
STRUCTURE:
HIV is a retro virus of class lenti-virus. RNA is the main genetic material. They also contain an
enzyme reverse transcriptase which upon entering into the host cells converts the RNA into DNA. The
4
RNA genetic material is surrounded by a capsid protein (p24). The outer layer consists of matrix protein
(P17) surrounded on the out by lipid bi-layer. The outer layer also consists of glycoprotein gp120 and
gp41 which helps in attaching the virus to the host cells (4, 7).
Figure 4 diagram of a HIV. Figure taken from “The EMBO J.
Vol.18 No.5 pp.1124–1136, 1999” (8)
5
Figure 5 Schematic diagram showing the entry mechanism of HIV. Figure taken from
S.J. Flint, L.W. Krug, V.R. Racaniello, A.M. Skalka- Principles of Virology- 2000
edition (7).
MECHANISM OF VIRUS ENTERING INTO CELLS:
The main receptor for HIV attachment on the host cell is CD4 which is present on T-cells and
macrophages. Along with the receptor, the virus also needs co-receptors  or  chemokine. The
interaction of the virus glyco-protein with chemokine triggers the fusion of virus and cellular
membranes and thus the virus gains entry into the cytoplasm. HIV-1 virus is further classified on basis
of the type of co-receptors it binds. -chemokine (Cxcr4) is present on T-cell hence the HIV is called Tcell-line-tropic strain of HIV-1 (X4) where as -chemokine (Ccr5) is present on macrophages so the
name given is macrophage-tropic strain of HIV-1 (R5) (4)(7).
HIV VACCINE:
The reason why there is a lot of problem in making a vaccine for AIDS is that HIV is a very
tricky virus and somehow avoids antibody binding. One of the tricks is that it has sugar molecules which
6
make the immune system believe that the virus is not foreign invader. Moreover the site with which it
binds to CD4 is hidden behind loops of proteins and carbohydrates, so it is very difficult to neutralize it.
On the other hand there is so much variation among different types of HIV that the antibody binding to
one will not bind to another (9).
The current trend in HIV vaccine research is by reverse immunology (which means designing a
vaccine or an immunoglobulin by studying antibodies that have been induced to that antigen (10)).
General Epitopes of the HIV-1 envelope glycoprotein along with the human monoclonal
antibodies are listed in table 1. Some of these examples will be discussed briefly. In one research
(11)
scientist solved the X-ray crystallographic structure of HIV gp120 in complex with CD4 receptor and a
neutralizing human antibody at 2.5 Angstrom resolution
Table 1 shows HIV epitopes and Human antibodies released against them. Data taken
from Nature march 2004 200-210 (10).
Epitopes
Human monoclonal Antibodies
Cluster I of gp41
Clone 3, 246-D
Transmembrane-proximal region of
gp41
2F5, 4E10, Z13
CD-4 binding domain of gp120
Ig G1b12, 559/64D, 15e
CD-4 induced epitope of gp120
17b,48D
1-2 mannose residues of gp120
2G12
V2 loop of gp120
697-D
V3 loop of gp120
447/52-D, 19b,2182
7
Methods for getting proteins: The gp120 used in the experiment was from HXBc2 stain of HIV-1.
They were produced from Drosophila Schneider 2 lines under the control of an inducible
metallothionein. Chinese hamster ovarian cells were used to produce the two domain CD4 (D1D2,
residues1-182). For monoclonal antibody 17b production, B-cell clone were used. B-cells were
immortalized by Epstein-Barr virus, isolated from an HIV-1 infected individual and were fused with
murine B-cell.
Strategy for crystallization: The biggest hurdle for the researchers was that gp120 had extensive
glycosylation and conformational heterogeneity. Hence the sample was deglycosylated and complexes
were formed with various ligands. Moreover they also made truncations at termini and variable loops in
various combinations. So 90% of carbohydrate was removed where as 80% of the non variable protein
was retained. Theoretically they believed that the probability of crystal formation is greatly increased by
such reduction of surface heterogeneity. This way they obtained crystals for ternary complex composed
of truncated gp120, N-terminal two domains (DID2) of CD4 and a Fab from the human neutralizing
monoclonal antibody 17b (see figure 6 below). A combination of techniques was used to solve the
ternary structure via molecular replacement, isomorphous replacement and density modification. The
two heavy atom compounds used were K3IrCl6 and K2OsCl6.
Figure 6 shows the overall X-ray crystallographic
structure of cd4, gp120 and fab 17b. Gp120 is in red, cd4
in yellow and fab 17b in light blue (light chain) and puple.
8
Figure is taken from Nature (1998) 393, 648–659 (11).
In a recent study
(12),
a similar interaction between gp120 and a broadly neutralizing human
antibody 2G12 was seen using X-ray crystallography (see figure 7 below). This antibody 2G12 was
isolated from patients infected with HIV. It is very special in the sense that it is the only
immunoglobulin isolated which successfully binds to carbohydrate part of glycoprotein. This
carbohydrate is the one which shields potential antigenic epitopes.
Figure 7 shows the x-ray crystallography
structure of gp120 and 2G12. Figure taken
from SCIENCE VOL 300 27 JUNE 2003 (12)
Another good research regarding structure and function of monoclonal antibodies can be seen
where scientist were trying to see the interaction of P24 (HIV capsid protein) with Fab13B5 antibody
(see figure 8 shown below) (13).
The importance of p24 is seen during virus assembly, maturation, and disassembly. Inside the
mature virions lipid bilayer, p24 forms characteristically conical shaped shell surrounding the RNAnucleoprotein complex. Antibody-antigen interactions are very important as their detection serves as
9
diagnostic for HIV infection. Moreover the antibodies presence in larger amount is correlated with
delayed progression to AIDS. Only 243 residues (called RH24) of p24 were considered for the research
and they were derived from HXB2 strain of HIV-1. X-ray crystallography structure of the antigen
antibody complex was determined in the previous research at 3 angstrom. In this research they only
developed the x-ray structure of the antibody at 1.8 angstrom resolution. The most fascinating part of the
research was that structural changes were observed between Fab bound to p24 and the free Fab as well
as p24 bound to Fab and free p24.
Figure 8 shows the interaction between P24 and the
antibody part Vh and Ch. Figure taken from Structure,
Vol. 8, 1069–1077, October, 2000(13)
In effort for AIDS vaccine development researchers were trying to trace the binding site of a
neutralizing monoclonal antibody 2F5 to HIV glycoprotein gp41 (14). The biophysical technique they use
was Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with
10
proteolytic protection assays. 2F5 is also among those antibodies which are isolated from HIV-1 infected
individuals. It can neutralize most primary HIV-1 isolates in vitro. Therefore it is of significant interest
to vaccine designers.
Monoclonal antibody 2F5 was obtained from the AIDS Research and reference reagent program.
The antihuman Fc-specific immunoglobulin secondary antibody was bought from Sigma chemical
company. The gp140 protein was expressed from the HIV-1 JR-FL env gene. This gene contains the
expression of gp120 linked to gp41 with disulfide linkage. The designated name of the protein is sos
gp140.
Under physiological conditions SOS gp140 was bound to 2F5 monoclonal antibody. Gp140
residues which were not protected by the antibody were cleaved using series of proteolytic enzymes (as
shown in figure 9 below).
Figure 9 shows a self explanatory diagrammatic
presentation of the experiment. Figure taken
from JOURNAL OF VIROLOGY, Nov. 2001,
p. 10906–10911 Vol. 75, No. 22 (14).
11
The 16 amino acid long sequence shown in the diagram is found to be the main epitope on gp41.
The research paper also mentions that, this sequence is significantly longer then the ELDKWA core
epitope previously determined for 2F5 by peptide ELISA. This shows that MALDI-MS is a better
method than ELISA. A typical mass spectrum of the experiment is shown below in figure 10.
Figure 10 shows a mass spectrum detecting the16 amino
acid chain residue. Figure taken from JOURNAL OF
VIROLOGY, Nov. 2001, p. 10906–10911 Vol. 75, No. 22
(14).
Another ground breaking research was reported by Susan Zolla-Pazner and co-scientist (8c). They
identified a neutralizing antibody known as 447-52D which binds to V3 loop of the glycoprotein gp120.
The structure of V3 was found to be similar to  hairpins present in chemokine co-receptors (CCR5 and
CXCR4) for HIV. All the structures were solved using NMR.
12
HIV needs to bind to chemokine co-receptors along with the main receptor CD4 in order to enter
the host cell. This indicates that mechanism stimulating antibodies similar to 447-52D might help in
developing a good vaccine against HIV.
Figure 11. The first figure on the top is the backbone
superimposition of 29 lowest-energy structure of V3 and the
second figure is the ribbon diagram of the energy minimized
average structure.
Figure taken from Structure, Vol. 11,
225–236, February, 2003 (15).
VACCINE DESIGN STRATEGIES:
Among the most successful way of vaccine design is the Salk’s polio vaccine method. It should
contain killed virus surface proteins eg in case of HIV, gp120, gp41 or p24. Next important one is
Sabin’s polio vaccine method using live vector viruses engineered to carry genes encoding HIV proteins.
A good example which is under clinical trial is where combination of elements such as pure gp120 and
canary pox vector are used (combination of both Salk and Sabin method). In some trials even naked
DNA is also been tried which code for one or more HIV genes. Live harmless bacteria engineered to
carry genes encoding HIV protein are also under trial. Some more examples are Pseudovirions (non-
13
replicating HIV-like particles) and Replicons (non-HIV viruses engineered to carry genes encoding HIV
proteins but they do not completely replicate) (16).
THE DISCOURAGEMENT:
Unlike Salk’s whole killed poliovirus HIV’s destroyed viruses couldn’t start immune reaction
because the structure was disrupted. A biotech company Genentech tried the clinical trial with only gp
120 but it was only Lab HIV specific and the HIV mutates very fast. Even Sabin’s live attenuated strains
of polio vaccine method also could not serve as a model because of the fear of active HIV getting in the
human body of volunteers (17).
CONCLUSION:
HIV is a tricky virus and it is creating a lot of problem. To solve the problem a multidisciplinary
research approach is a must. Identification of successful antibodies like 2G12 and 2F5 and their atomic
level structure determination to study the interaction is a big success in keeping the hope up. There is no
doubt that physical techniques like X-ray, NMR and spectroscopy are bringing us closer to understand
our limitation in making an AIDS vaccine but still there is a long wait before a success story will be
made.
REFERENCES:
(1) http://www.pbs.org/wgbh/aso/databank/entries/bmsalk.html
(2) Carl Branden, John Tooze-Introduction to protein structure- second edition.
(3) http://cancerweb.ncl.ac.uk/cgi-bin/omd?query=epitopes&action=Search+OMD
(4) David M. Knipe, Peter M. Howley- Fundamental Virology-Fourth Edition.
(5) http://www.whfreeman.com/immunology/CH05/kuby05.htm
14
(6)
http://www.whfreeman.com/immunology/CH02/hybridomas.htm
(please try Google or cut and paste in web
browser)
(7) S.J. Flint, L.W. Krug, V.R. Racaniello, A.M. Skalka- Principles of Virology- 2000 edition.
(8) Carmen Berthet-Colominas, Ste´ phanie Monaco, Armelle Novelli1, Genevie`ve Sibaı¨2, Franc¸ois
Mallet1 and Stephen Cusack3--Head-to-tail dimers and interdomain flexibility revealed by the
crystal structure of HIV-1 capsid protein (p24) complexed with a monoclonal antibody Fab-- The
EMBO Journal Vol.18 No.5 pp.1124–1136, 1999
http://www.nature.com/cgi-taf/DynaPage.taf?file=/emboj/journal/v18/n5/full/7591546a.html&filetype=pdf
(9) Review Michael Hortens
The Quest for Neutralizing Antibodies to HIV
A I D S VA C C I N E A D V O C A C Y C O A L I T I O N • WWW. AVAC.ORG
http://avac.org.phtemp.com/pdf/reports/AbResearch.pdf
(10)
Review
Susan Zolla-Pazner- IDENTIFYING EPITOPES OF HIV-1
THAT INDUCE PROTECTIVE ANTIBODIES- Nature march 2004 200-210.
http://www.nature.com/nri/journal/v4/n3/pdf/nri1307.pdf
(11) Article
Hendrickson,
Kwong, P.D., Wyatt, R., Robinson, J., Sweet, R.W., Sodroski, J., and
W.A. (1998)-- Structure of an HIV gp120 envelope glycoprotein in complex with
the CD4 receptor and a neutralizing human antibody --Nature 393, 648–659.
http://www.nature.com/cgitaf/DynaPage.taf?file=/nature/journal/v393/n6686/full/393648a0_fs.html&content_filetype=pdf
(12) Daniel A. Calarese, Christopher N. Scanlan, Michael B. Zwick, Songpon Deechongkit, Yusuke
Mimura, Renate Kunert, Ping Zhu, Mark R. Wormald, Robyn L. Stanfield, Kenneth H. Roux,
Jeffery W. Kelly, Pauline M. Rudd, Raymond A. Dwek, Hermann Katinger, Dennis R. Burton,
15
Ian A. Wilson,-Antibody Domain Exchange Is an Immunological Solution to Carbohydrate
Cluster Recognition SCIENCE VOL 300 27 JUNE 2003
http://medicine.ucsd.edu/molpath/PDF/Wilson_CelBiol.pdf
(13) Ste´phanie Monaco-Malbet, k Carmen Berthet-Colominas, Armelle Novelli, Nicole Battaı,
Nadia
Piga, Vale´ rie Cheynet,† Franc¸ ois Mallet, and Stephen Cusack-- Mutual
Conformational Adaptations in Antigen and Antibody upon Complex Formation between an Fab
and HIV-1
Capsid
Protein
p24
Structure,
Vol.
8,
1069–1077,
October,
2
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VSR-41M94YH-7G&_cdi=6269&_user=585204&_orig=search&_coverDate=10%2F31%2F2000&_qd=1&_sk=999919989&view=c&
wchp=dGLbVtz-zSkzS&md5=36f4c2d713c65410a03bef4d8552d7bc&ie=/sdarticle.pdf
(14) CAROL E. PARKER,1* LEESA J. DETERDING,1* CHRISTINE HAGER-BRAUN,1 JAMES
M. BINLEY,2 NORBERT SCHU¨ LKE,3 HERMANN KATINGER,4 JOHN P. MOORE,2
AND KENNETH B. TOMER1 Fine Definition of the Epitope on the gp41 Glycoprotein of
Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5-JOURNAL
OF VIROLOGY, Nov. 2001, p. 10906–10911 Vol. 75, No. 22
http://jvi.asm.org/cgi/reprint/75/22/10906
(15) Michal Sharon,1 Naama Kessler,1 Rina Levy,1 Susan Zolla-Pazner,2 Matthias Go¨ rlach,3 and
Jacob Anglister--Alternative Conformations of HIV-1 V3 Loops Mimic _ Hairpins in
Chemokines, Suggesting a Mechanism for Coreceptor Selectivity --Structure, Vol. 11, 225–236,
February, 2003
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VSR-47WC25R-GF&_cdi=6269&_user=585204&_orig=browse&_coverDate=02%2F28%2F2003&_sk=999889997&view=c&wchp
=dGLbVzz-zSkzV&md5=d3bbc4b5da7f2b46e84c700b1203fa63&ie=/sdarticle.pdf
16
(16) http://www.niaid.nih.gov/daids/vaccine/slidehivvacupdate/slide15.htm
(17) http://pubs.acs.org/cen/topstory/7935/7935sci1.html
17