HIV-1 specific humoral immune responses in
healthy volunteers after HIV DNA prime - HIV
MVA boost immunization
Sarah Petersson
Degree project in biology, Master of Science
2010
Biology Education Center, Uppsala University and
Swedish Institute for Infectious Disease Control
Karolinska Institutet Stockholm supervisors
Andreas Bråve, Jorma Hinkula, Britta Wahren
2
SUMMARY
Since the discovery of HIV-1, more than 60 million individuals have been infected and 3
millions are infected every year. Several antiviral drugs are available that greatly suppress the
viral replication and prolongs the life of the infected individual, but the drugs cannot cure the
host from the virus. In order to stop the HIV-epidemic there is an urgent need of a vaccine.
However, so far all traditional vaccine strategies have shown to be inefficient (and potentially
dangerous) and new vaccine approaches are being explored. Our group has designed a genetic
vaccine encoding different parts of the HIV-1 virus. The HIV-1 genes are carried by DNA
plasmids and by the viral vector, modified vaccinia virus Ankara (MVA). In the first phase I
trial with the vaccine, 40 Swedish, healthy volunteers were immunized by three priming
plasmid DNA immunizations and 6 months later boosted once with the MVA. Several years
later, 24 of the volunteers received a second boost with MVA. The result after the 1st MVA
immunization was highly successful and all but one volunteer responded with HIV-1 specific
immune responses. In this project, the humoral responses were investigated against different
HIV-1 structures: capsid/matrix (Gag), envelope (Env) and the enzyme reverse transcriptase
(RT), after the 2nd MVA boost. The result showed that a 2nd boost of MVA was required in
order to elicit detectable Env-specific antibodies. Gag-specific antibodies were relatively high
already after the 1st MVA boost, but increased after the 2nd. A few vaccinees developed a
response against RT. The volunteers were vaccinated differently in order to investigate which
delivery method that would be preferred for the vaccine. The volunteers were either
immunized with different amounts of vaccine intradermally or intramuscularly and with or
without the adjuvant GM-CSF. Due to low numbers of individuals in each group, no clear
conclusion could be made regarding the best delivery strategy.
The results presented here show that the vaccine can induce Env- and Gag-specific
antibodies, in addition to the already reported cellular responses, and in addition to an
ongoing phase I trial in Tanzania, the vaccine will shortly enter a new phase I trial in Sweden
as well as two phase 2 trials in Tanzania and Mozambique.
3
INTRODUCTION
Human immunodeficiency virus (HIV)
In the early 80´s several cases of diseases, which normally only occur in immunodeficient
persons, were reported among homosexual men in the US. The patients’ immune systems and
especially the CD4+ T lymphocytes were depleted, and this new disease was given the name
acquired immunodeficiency syndrome (AIDS). In 1983, the French scientists Francoise
Barré-Sinoussi and Luc Montagnier isolated a novel retrovirus from the lymph node of an
AIDS patient and the virus was identified as the causative agent of AIDS [4, 33, 44]. The
retrovirus received its name, HIV, in 1985 [37] and the French scientists Montagnier and
Barré-Sinoussi were honoured with the Nobel Prize in 2008 for their discovery.
Since the start of the epidemic, approximately 60 million people have been infected with
HIV-1 and today, 33 million individuals live with the disease, 3 millions are newly infected
and 2 millions die every year due to AIDS-related diseases [67].
The virus can be divided into two types; HIV-1 and HIV-2. Both originate from African
simian immunodeficiency viruses (SIVs) that infect non-human primates and according to
phylogenetic studies, the jump from monkeys to humans is believed to have occurred during
the early 20th century [28]. HIV-1, which is the cause of the world wide epidemic, is believed
to originate from the chimpanzee SIV (SIVCPZ). The HIV-2, which is mainly found in
Western Africa is believed to originate from sooty mangabey SIV (SIVSM) [21, 56]. HIV-1
can further be divided into three different groups: M (main), O (outlier) and N (non-M nonO), where M is most common. Viruses within group M can be further divided into subtypes
(or clades) A-K, which are geographically distributed. The subtype B virus mainly circulates
in the western part of the world [47], whereas subtype C is the most common subtype in the
world and is mainly found in southern Africa and India [67].
Structure and replication
HIV-1 is a Lentivirus, a genus within the Retroviridae family. It is a spherical, enveloped
virus with a diameter of 110 nm. The genome is approximately 10 kB and consists of two
positive single stranded RNA molecules, which contain nine genes. Like other retroviruses,
HIV-1 has the genes env, gag and pol. In addition, HIV-1 carries regulatory genes:
transactivator of viral transcription (tat), regulator of RNA transport (rev); and accessory
genes: viral infectivity factor (vif), viral protein R (vpr), viral protein U (vpu) and the negative
factor (nef) [18].
The envelope consists of a lipid bilayer that is obtained from the host cell during budding
and within the membrane the viral glycoproteins gp120 and gp41 protruds (Figure 1).
Trimeric gp120 is non-covalently associated with trimers of the transmembrane protein gp41.
The two proteins derive from the polyprotein gp160, which is encoded by the viral env-gene
[31]. The capsid (p24) and the nucleocapsid (p7) build up the viral core and they protect and
encapsulate the viral genome. The nucleocapsid is in turn surrounded by the matrix protein
p17, which stabilizes the virus. These three proteins derive from the polyprotein p55Gag that
is encoded by the gag-gene. From the pol-gene, the three viral enzymes are transcribed and
translated; reverse transcriptase (RT), protease (PR) and integrase (IN) (Figure 1) [31].
4
gp120 gp41 p17 p24 p7 RNA RT Figure 1. HIV-1 structure. The virus consists of different proteins: This picture includes gp120 and gp41
deriving from env; p17, p24 and p7 encoded from gag and RT from pol. The genome is composed of two
single-stranded RNA molecules.
Infection begins when the gp120 envelope protein binds to the CD4 molecule on the target
cells like T cells, monocytes and dendritic cells (DCs) (Figure 2). This causes conformational
change of the protein, allowing for the gp120 to bind to the chemokine co-receptors CXCR4
and CCR5. This interaction brings the virus closer to the cell and allows for the gp41 protein
to penetrate the cell membrane resulting in fusion of the viral with the cellular membrane.
When it enters the cytoplasm, the virus´ own RT turns single stranded (ss) RNA to double
stranded (ds) DNA which later is integrated into the host cell genome by the enzyme IN. The
RT enzyme does not have the capacity of proof-reading (as cellular polymerases) and that
leads to a high mutation frequency [18, 62]. Some of the HIV-1 genes are first transcribed and
translated into precursor proteins, which enhance the transcription and translation of the late
structural genes. The gp160 protein is glycosylated in the endoplasmic reticulum (ER) and
cleaved into gp120 and gp41 in the golgi apparatus. The proteins are later assembled by the
cell membrane. The pol and gag genes are transcribed and translated into the polyproteins
p55Gag and p160GagPol. The newly formed proteins are cleaved and assembled at the cell
membrane. Thereafter, new viruses bud from the surface (Figure 2) [19].
5
1
CD4
CCR5, CXCR4
2
3
T cell
4
5
6
Figure 2. HIV-1 replication. (1) Replication starts when the virus binds to the CD4 molecule and the co-receptor
(CCR5, CXCR4) and fuses with the cell membrane. (2) In the cytoplasm, the capsid is released and the viral
RNA is transcribed into DNA by its own RT. (3) The viral DNA is later transported into the nucleus and
integrated into the host genome. (4) Once integrated, the viral genome is transcribed by the cellular transcription
machinery, and the transcripts are translated in the cells cytoplasm into precursor proteins (5), which are cleaved
during assembling and budding (6) of the virus.
6
Infection and immunology
HIV-1 can be transmitted through several routes, but the most common is through unsafe
vaginal or anal sex. Other routes are by transfer of blood (using unclean needles, during
transplantation or transfusion and from mother to child (during pregnancy, birth or
breastfeeding). The risk of acquiring HIV-1 infection during vaginal intercourse is relatively
low, approximately one out of 100 exposures, but the risk depends on viral load of the
infected partner and presence of other genital infections [36, 65].
The infection occurs when the virus enters the blood stream or is transmitted over a
mucosal surface. Here, the virus either directly infects or attaches to antigen presenting cells
(APCs) such as DCs or macrophages (MØ), but the virus can infect any cell that expresses
both the primary receptor CD4 and any of the co-receptors CCR5 or CXCR4. DCs transport
the virus to the lymph nodes, where it can infect T lymphocytes. The virus has a preference
for infecting CD4+ T cells (especially memory T cells) and the initial infection is massive
with high viral replication. This stage is called the acute phase, which can last for 2-3 months
and often manifests as flulike symptoms [27]. The cell-mediated immune response against
HIV-1 consists of activated CD4+ T cells that help CD8+ T cells to mature to cytotoxic T cells
(CTLs). The CTLs kill HIV-1 infected cells and suppress the virus. They also secrete different
chemokines (RANTES, MIP-1α/β) which have a protecting function against the HIV-1 virus.
The viral load decreases to a stable level termed the viral set-point and is a good indicator of
disease progression. The patient now enters the chronic phase that can last for several years
(16).
The humoral immunity includes B cells and their production of antibodies (abs), which are
located on or secreted by different types of B cells. Anti-HIV ab can be found a few weeks
after infection and most of the abs are directed to the viral envelope proteins. The majority of
produced abs often are non-neutralizing (they cannot neutralize the virus) and the
development of neutralizing abs takes several months [68].
As the infection progresses, the CD4+ T cells are eliminated, and when the CD4 T cell
count reaches below 200 cells/mm3 blood the patient develop AIDS and opportunistic
infections appear (Figure 3) [16, 22]. The early destruction of memory T cells is believed to
play a substantial role in the progress to AIDS.
CD4+ T cells per mm3 Viral load 1000
107
Latency 106
105
500
104
103
200
102
Weeks Years Months Figure 3. The clinical course of HIV-1 infection. When infection occurs, the CD4+ T cell number drops and the
viral
load increases. This stage lasts for weeks and is called the acute phase. The next phase is a latency stage and
at this stage, the viral number decreases to a stable level (set-point). CD4+ T cell number declines during this
AIDS.
stage and when reaching 200 cells/mm3, the disease is called
7
HIV-1 treatment
Despite efforts, HIV-1 is still spreading and for every two people starting treatment, another
five are newly infected [67].
The first promising drug, azidothymidine (AZT), was licensed 1989 but as we know today
monotherapy against HIV-1 is inefficient due to the high mutation rate of the virus. To drugs
targeting different parts of the virus is essential this type of therapy is called Highly Active
Antiretroviral Therapy (HAART). The most common drugs target RT and protesase but novel
drugs have recently been licences that target other steps in the viral life cycle, such as
integrase inhibitors and chemokine inhibitors [43]. HAART does not cure the infected
individual but it can very efficiently reduce the viral load and increase the life expectancy and
the quality of life for the infected person. Since the introduction of HAART, the overall
mortality and viral spread have decreased significantly, especially in the developed world.
Yet, the virus can survive, replicate and mutate despite all the drugs. The drugs are expensive
and thus non-available in poor countries in an adequate extent. [26].
Vaccines against HIV-1
Vaccination is a process where the immune system is instructed to combat a certain pathogen
(or tumor). A vaccine can be achieved in different ways and forms with the aim to produce a
long-lived pathogen-specific immunity. The adaptive cellular immune response consists of
CD4+ and CD8+ T cells. The humoral response is composed of B cells that generate
neutralizing antibodies (Nab), which can directly inhibit the pathogen. When a vaccinated
individual is exposed to the pathogen, the immune system is reactivated and able to clear the
infected agent faster than if the individual is unvaccinated. Compared to therapeutic treatment
vaccination is highly cost-effective [20, 38]. In the case of HIV-1, so far all the traditional
vaccine strategies have been unsuccessful and all HIV-1 vaccine candidates have failed in
clinical trials [3, 20]. Resistance against HIV-1 infections exist in some healthy individuals. A
number of people lack the co-receptor CCR5, due to a deletion in the CCR5 gene, which
makes them immune to the virus [53]. Also in some HIV-1 infected persons, the infection can
be controlled without treatment. They often have a powerful cell-mediated and humoral
immunity [51, 6]. Non-Nabs are abundant in all HIV-1 infected individuals, but these nonNabs do not kill the virus. However, some infected persons develop Nabs several months after
infection during the latency stage. It has been shown that Nabs have a protective function and
can reduce the progression towards AIDS [42, 68].
Live attenuated virus vaccines
Live attenuated virus vaccines contain replicating virus that have undergone a gradient loss of
virulens, meaning that they no longer cause disease. The great advantage of this type of
vaccines is that it elicits both strong cell-mediated and humoral immunity. This strategy is
considered too dangerous for HIV-1. This was evident in a cohort of patients in Australia,
where patients by mistake were given blood containing nef-deficient HIV-1. Initially, the
patients were able to control the viremia and they did not progress to AIDS. However, the
virus was able to revert to a pathogenic form and ultimately caused clinical disease and AIDS
[32]. Moreover, in a non human primate study, a nef and vpr deficient SIV vaccine was
evaluated but also here the virus reverted and caused the monkeys to develop AIDS [1].
8
Inactivated virus vaccines
In inactivated vaccines, the virus is inactivated, either chemically or by heat, before
administration. It induces strong humoral responses, but only weak cell-mediated protection.
The use of inactivated viruses has not shown to be protective in any study, but can be utilized
ex-vivo as antigens to monitor HIV-1 responses [20, 34].
Subunit vaccines
Subunit vaccines are composed of proteins or peptides, which have been synthesized from
HIV-1. Peptides are easier to design and produce, but are less immunogenic than proteins.
Subunit vaccines induce mainly strong antibody (ab) response [20].
Protein vaccines have been extensively evaluated against HIV-1, especially vaccines
containing the monomeric gp120 protein. In several studies, increasing CD4+ T cells and
neutralising antibodies (Nabs) against laboratory adapted HIV-1 strains were induced (35,
55). A larger clinical trial with a recombinant gp120 subunit vaccine including subtype B and
E (AIDSVAX) was performed by the company Vaxgen, but the vaccine showed no protection
from infection or reduction in viral load after infection [14, 17].
Genetic vaccines
In the beginning of the 1990s, it was shown that direct delivery of influenza genes into mice
could confer protection from subsequent viral challenge [63]. So called naked genetic
vaccines contain DNA or RNA from infectious agents. In the case of naked DNA vaccines,
the genes are often delivered by a stable bacterial plasmid. The delivery unit is taken up by
various cells, which transcribes and translates the gene into protein. Genetic immunization has
some advantages over conventional vaccines. It can both induce humoral and cell-mediated
immunity, it is very safe and the plasmids and insert can easily be constructed and adjusted
[20, 29, 64].
Viral vectors
To enhance the immune response of DNA vaccines, viral vectors are often used for boosting
responses [9]. There are viral vectors based on DNA and RNA viruses, but the most
frequently used are various adenoviruses and attenuated pox-viruses such as ALVAC [61]
NYVAC [24] and modified vaccinia virus Ankara (MVA) [40]. Because the Ad5 virus is
prevalent in the human population, problems with anti-vector immunity arose and in the fall
of 2007, a large trial failed [10, 39]. The focus now lies on modified Ad5 viruses or other, less
immunogenic Adenoviruses [13].
In this study, we use the MVA as a vector vehicle. This virus belongs to the
orthopoxviruses and contains a large and stable double stranded DNA genome. An advantage
is that the MVA has fewer problems with anti-vector immunity than many other viral vectors
[7, 23, 54, 59, 60].
Trials of genetic vaccines
In the first human clinical trial, DNA plasmids encoding Nef, Rev, Tat and Env were
administrated into HIV-1 infected individuals. The vaccine was able to induce novel T cell
responses [12]. DNA-priming can prime for an ab response of better quality than other
vaccine modalities. This effect can be explained by that the expressed protein is correctly
9
folded and presented as the process is mimicking what is happening during a native infection.
Together with viral vector boosting, which induces strong T cell responses that also resembles
what happens during natural infections, this HIV-1 vaccine strategy has become very popular.
The potential and capacity of prime-boost immunizations were first discovered in one study
with the non-human macaques, which were primed with vaccinia virus expressing SIV gp160
and then boosted with recombinant SIV gp160 produced in baculo-virus infected cells [25].
A number of larger clinical trials have been performed and evaluated. After extensive
research, a canarypox-based vector (ALVAC) encoding HIV-1 gp120 Gag, fragments of Gag
and Pol were developed and evaluated in clinical trials, 2007. It induced both cellular and
humoral responses, but the responses were overall low both for CD8+ T cells responses and
abs [52]. Recently a larger clinical trial was completed. It was carried out on a large number
of volunteers in Thailand, who were administered with both the viral vector ALVAC and the
subunit vaccine AIDSVAX (recombinant gp120). The results were promising with a vaccine
efficacy of 30 % [46]. Another promising vaccine trial that got a lot of attention was the STEP
study. The trial included adenovirus 5 vectors encoding HIV-1 Gag, Pol and Nef. It was
administrated several times in 3000 non-infected participants, but the vaccine did not prevent
the infection or lower the viral loads [10].
Enhancing vaccine potency
Adjuvants
A problem with genetic HIV-1 vaccines is that they are less immunogenic, especially in larger
animals, than the more classical vaccines. Thus, there is a need for compounds that can
augment vaccine potency and help to induce a strong and long-lived immune response. These
compounds are called adjuvants and have been used in vaccination since early 1920s. With
these enhancers, one can also direct the immune system towards a specific type of response.
Moreover, the amount of antigen used and number of immunizations can be decreased if a
proper adjuvant is used. The first adjuvant used was ALUM, an alum hydroxide/phosphate
salt that is still employed today [41].
Our group has used the recombinant granulocyte macrophage colony-stimulating factor
(rGM-CSF) as an adjuvant. This cytokine is produced by a number of cells including
macrophages, activated T cells and mast cells, and the protein stimulates stem cells to produce
white blood cells like granulocytes and macrophages and also stimulates professional APCs to
mature. As an adjuvant, it has shown to increase T- cell and ab responses in mice and nonhuman primates [8, 48, 49], but in humans the results have not looked good. Delivered with
either HIV-1 peptides or HIV-1 DNA vaccine, the GM-CSF does not affect nor has even a
negative effect on the immune response [54].
Today, only a few adjuvants are licensed for human use and include alum and certain oilin-water emulsion adjuvants [41].
DNA vaccine delivery methods
A DNA-vaccine can be administrated in different ways. The first delivery methods used were
needles and syringes and the gene gun. The latter induces cellular but predominantly humoral
immune responses and the DNA, which is coated onto gold beads, is delivered by heliumpropelled acceleration. Lately, novel delivery techniques have been invented and one used in
our clinical vaccine trials is the Biojector. This CO2-propelled device is needle-free and the
DNA is injected to target cells with the aid of a sturdy liquid flow. However, the delivery
method which has shown most potential so far is electroporation [57]. Here, the DNA is
10
transferred to cells by electric pulses that subvert cellular membranes. The technique has
shown an increased breath in immune responses and will likely be used in future clinical trials
[50, 15].
HIVIS
The HIVIS trials are a collaboration between the Swedish Institute for Infectious Disease
Control, Karolinska Institute, the Muhimbili University College of Health Sciences in
Tanzania and the Walter Reed Army Institute for Research in the US. The phase 1 clinical
trial comprises healthy volunteers from both Sweden and Tanzania, who have been
administered a HIV-1 vaccine consisting of DNA prime and MVA boost. The vaccine contain
seven different plasmids encoding HIV-1 Env subtype A, B and C, Gag subtype A and B, Rev
and RT subtype B. The viral vector MVA has been developed by the US National Institutes of
Health and the Walter Reed Army Institute of Research and contains HIV-1 Env subtype E
and Gag/Pol subtype A [8] (Figure 4).
A
B
Figure 4. The HIVIS-vaccine. (A) Plasmid-vectored HIV-1 containing genes and (B) the viral vector
modified vaccinia virus Ankara. Design of figures: Dr. Karl Ljungberg and Dr. Patricia Earl.
In the Swedish trial, 40 healthy volunteers between the ages 18-40, male and female, were
divided into four different treatment groups. They were first primed with different amounts of
DNA-vaccine by Biojector injection on days 0, 30 and 90 and two groups also received the
GM-CSF adjuvant either by intradermal (ID) needle injection or intramuscular (IM) Biojector
injection. Six months after the last DNA injection, the volunteers were given either a single
ID HIV-1 MVA injection of 107 plack forming units (pfu) or IM vaccination of 108 pfu HIV-1
MVA (Figure 5). The aim of the HIVIS trials in Sweden was to study safety and
immunogenicity before designing a similar trial in Tanzania. The results were successful with
more than 97% HIV-1 specific cellular immune responders. Yet, only a few developed abs
against HIV-1 [54].
A 2nd second MVA boost was administrated as 108 pfu IM into 24 of the volunteers during
summer 2009 (Figure 5).
11
Figure 5. Immunization schedule. DNA was injected in 40 volunteers at days 0, 30 and 90 either
intradermally (ID) or intramuscularly (IM). Six months after the last DNA injection, the volunteers were given
either a single ID HIV-1 MVA injection of 107 plack forming units (pfu) or IM vaccination of 108 pfu HIV-1
MVA. A 2nd MVA boost was administrated as 108 pfu IM into 24 volunteers three years after the 1st MVA
boost. Immune respons tests were performed two and forty weeks after the 1st MVA and two and four weeks
after the 2nd MVA boost.
AIMS This study had the following aims:
-
To detect the amount of (titer) IgG abs against Gag, Env and RT after a second boost
of MVA in 23 healthy volunteers.
To detect epitope-specific abs against gp41 after a second boost of MVA in 23 healthy
volunteers
12
MATERIALS AND METHODS
ELISA
Indirect ELISA was carried out on plasma from 24 Swedish, participating in the HIVISvaccine trial. The group of volunteers consisted of male and female between the ages 18-40.
Ninety-six well ELISA plates (Nunc Maxisorp, Odense, Denmark) were coated with gp160
(1.0 ug/mL, MicroGenesys Inc, Meriden CT, USA), recombinant p17/p24 (1.5 ug/mL, FIT
Biotech, Tartu, Estonia), RT (1.0 ug/mL, Centre for AIDS reagents (CFAR), NIBSCH, Herts,
UK) or gp41 peptides (10 ug/mL, Thermo Hybaid D-89077 Ulm Germany) from HIV-1
subtype B in 0.05 M NaCO3 pH 9.6. Plates were incubated over night in room temperature
and then stored at 4oC until further use. The ELISA-plates were washed three times with 0.9
% NaCl (Merck, Darmstadt, Germany) supplemented with 0.05 % Tween 20 (Sigma-Aldrich,
Stockholm, Sweden). Plates were then blocked with 5 % fat-free milk (Semper, Sweden) in
phosphate buffered saline (PBS) for one hour at 37oC. Plasma samples were diluted in 2.5 %
fat-free milk in PBS, added to the plates and incubated over night at 4oC. Plates were then
washed four times and rabbit anti-human IgG abs conjugated to horse radish peroxidase
(HRP) (DAKO P0214, Glostrup, Denmark), diluted 1:5000 in 1.25 % fat-free milk in PBS
was added. Subsequently, the plates were washed and O-phenylenediamine dihydrochloride
tablets in 0.1 M NaCitrate buffer were activated by 0.3 % H2O2 (Sigma-Aldrich, Stockholm,
Sweden) and added for development, 15-30 minutes for colour reaction. The reaction was
stopped with 2.5 M H2SO4 and the optical density (OD) was read at 490 nm using a
spectrophotometer.
Table 1. ELISA set up. ELISA plates were coated with the HIV-1 Gag, Env and RT antigens. Plasma
from 23 immunized volunteers was analyzed from different time-points and in different dilutions.
Antigen and plasma dilution
Env 1:100 1:200 1:400 1:800 1:1600
Gag 1:50 1:250 1:1250 1:6250 1:31250
RT 1:40 1:80 1:160 1:320
Baseline 2w post 1 MVA 40 w post 1 MVA 2 w post 2 MVA 4 w post 2 MVA
x
x
x
x
x
x
x
x
x
Statistical analysis
Laboratory data were entered and analyzed in Microsoft Excel. The serological cut-off was
calculated as the mean OD plus three standard deviations (SD) of the negative human
samples. The endpoint titer was defined as the point where the plasma dilution drops below
the cut-off. Statistical analyzes were performed using the GraphPad Prism 5.0 software.
Comparison between means of two groups was performed by the non-parametric MannWhitney U test. A P-value of < 0.05 was considered significant.
13
RESULTS
HIV-1 specific abs were found in all immunized volunteers
HIV-1 specific IgG abs were detected by using indirect ELISA. Blood plasma collected at
different time-points during the immunization protocol (baseline, 2 weeks post 2 MVA, 4
weeks post 2nd MVA) from 23 healthy, immunized volunteers was screened against three
antigens from HIV-1: gp160Env, p24/p17Gag and RT. Originally, 24 volunteers underwent
the whole immunization and received their 2nd boost with MVA, but one sample was excluded
from the analysis due to lack of baseline sample. The end-point titer, which is the highest
sample dilution that shows positive reactivity, was determined for each sample from each
individual. The cut-off for positive reactivity was calculated as the mean optical density (OD)
plus three standard deviations (SD) of the negative human samples from all the individuals.
After the 2nd MVA immunization, all volunteers developed abs against Env and Gag, but
only a few responded to RT. The volunteers responded the strongest against Gag, both after
the 1st and 2nd MVA (Appendix 1).
a
p = <0.0001
p = <0.0001
b
p = <0.0001
p = <0.0001
p = <0.0001
c
p = 0.0149
p = 0.9103
Figure
6.6.
End
point titers
ofof
HIV-1
specific
antibodies
found
inhealthy,
23 healthy,
Swedish
volunteers
immunized
with
a HIV-1DNA
DNA
Figure
Endpoint
titers
HIV-1
specific
abs found
in 23
Swedish
volunteers
immunized
with
a HIV-1
nd2nd boost of MVA (n = 23).
prime
-MVA-boost
vaccine.
(a)
Antibodies
against
env
were
detected
with
ELISA
after
a
prime-MVA boost vaccine. (a) Abs against env were detected with ELISA after a 2 boost of MVA (n = 23). (b) Abs
( b) Antibodies directed towards gag were identified already
after a 1st MVA boost, (n = 11) but the number of antibodies increased
directed towards gag were identified already after a 1st MVA boost, (n = 11) but the number of abs increased after a 2nd MVA
after a 2nd MVA boost. (n = 23). (c) Several volunteers developed antiibodies
againt RT after a 2nd MVA boost (n = 10), but a few
boost (n = 23). (c) Several volunteers developed abs against RT after a 2nd MVA boost (n = 10), but a few already had RT ab
aldready had RT antibody responses before MVA-boost (n = 3). The criterion for statistical significance was p < 0.05.
responses before MVA-boost (n = 3). The criterion for statistical significance was p < 0.05.
14
Env abs were not found after the 1st MVA immunization, but were readily detected after the
2nd MVA boost (Figure 6a) The end-point titer values after the 2nd MVA varied from 22 to
1723 in the volunteers. No difference was observed between titers at 2 weeks and 4 weeks
after the 2nd MVA, but the reactivity was significantly higher (p < 0.0001) than both baseline
reactivity and reactivity after the 1st MVA values. Half (12/23) of the individuals developed
abs against Gag already after the 1st MVA boost and this was considered significant as
compared to the baseline value (p < 0.0001) (Figure 6b). The 2nd MVA immunization
significantly induced and increased end-point titers of Gag abs in all volunteers (p < 0.0001),
resulting in end-point values varying from 165 to 8566. As was observed for the Env
responses, no difference in Gag responses was seen between samples from 2 weeks and 4
weeks post 2nd MVA (Figure 6). The increase in ab-response against Env and Gag over the
course of immunization protocol can be observed in figure 7a and b. Interestingly, one
individual shows an opposite trend, with a decreasing titer against Gag after the second MVA
boost (volunteer 12, depicted as a yellow line in Figure 7b). The 2nd MVA boost induced RTspecific ab titers in 10 out of 23 individuals and the titers after the 2nd MVA were significantly
higher (p = 0.0149) than baseline values and values after the 1st MVA boost (Figure 6c). The
RT-specific end-point titers after the 2nd MVA varied from 41 to 839. Three volunteers had
anti-RT abs already in their baseline samples. Volunteers who developed RT abs showed an
analogous trend as Env and Gag responses when RT ab responses were plotted over time
(Figure 7c). Figure 8 shows the correlation between Gag and Env ab titers.
a
b
c
Figure
Figure
7. HIV-1
specific
ab responses
time inover
23 healthy,
Swedish
volunteers.
(a) Env ab titers
overantibody
time, showing
7. HIV-1
specific
antibody over
responses
time in 23
healthy,
Swedish volunteers.
(a) Env
nd
nd
ab response
after
a
2
MVA
boost
(n
=
23).
(b)
Gag
ab
levels
increased
after
a
2
boost
of
MVA
(n
=
23).
(c)
A number of
titers over time, showing antibody response after a 2nd MVA boost (n = 23) (b) Gag antibody levels increased
nd
after
a
2nd
boost
of
MVA
(n
=
23).
(c)
A
number
of
(n
=
10)
volunteers
had
increased
RT
response
after
a 2nd
MVA
boost.
volunteers
(n
=
10)
had
increased
RT
response
after
a
2
MVA-boost.
15
p = 0.0109
Figure8.8.Correlation
Correlationbetween
between HIV-1
HIV-1 env
antibody
response
in 23
Figure
Envand
andgag
Gag
ab responses
in 23
healthy,
healthy, Swedish volunteers. A correlation analysis demonstrated significant
Swedish volunteers. A correlation analysis demonstrated significant correlation
correlation (0.520) (p = 0.0109). The criterion for statistical significance was p <
(0.520)
0.05. (p = 0.0109). The criterion for statistical significance was p > 0.05.
Responses according to treatment
The twenty-three volunteers were randomized into 4 groups, which were immunized
according to different protocols. They received the DNA plasmids either ID or in IM, with or
without the adjuvant GM-CSF. The individuals were subsequently divided further to receive
the 1st MVA boost either ID or IM. To determine which delivery route that would be preferred
in a future vaccine, the end-point ab titers were compared for the different groups. Individuals
immunized with DNA IM developed slightly better Gag ab titers than volunteers receiving
DNA ID, but the difference was not significant (p = 0.2045) (Figure 9a). Moreover,
volunteers receiving the 1st MVA boost IM had higher Gag-specific ab titers than individuals
immunized ID (p = 0.0256). There was no difference in env ab titers when comparing IM or
ID injection (Figure 9b). Volunteers with the highest ab responses against both Env and Gag
were immunized with DNA and MVA IM without any adjuvant (Figure 9).
a
p = 0.2045
p = 0.0256
b
Figure9.
9. Group
Group specific
response
23 healthy,
Swedish
volunteers.
Volunteersreceiving
receivingthe
theDNA
DNA prime
prime IM
Figure
specificgag
Gagantibody
ab response
in 23inhealthy,
Swedish
volunteers.
(a)(a)
Volunteers
IM
developed a better gag antibody response than DNA ID, but was not considered significant (0.2045). Volunteers immunized
developed a better Gag ab response than DNA ID, but was not considered significant (0.2045). Volunteers immunized with MVA
with MVA IM had better gag antibody titers than individuals receinving the MVA ID (p = 0.0256). (b) No difference in immunization
IM
had better Gag ab titers than individuals receiving the MVA ID (p = 0.0256). (b) No difference in immunization delivery
delivery strategy was seen when analyzing env antibody titers. Black spots represent volunteers who not received the adjuvant GM-CSF.
strategy
wasare
seen
when analyzing
Envwith
ab titers.
Black spots
represent volunteers
who did notThe
receive
the for
adjuvant
G-CSF. Purple
Purple dots
volunteers
immunized
the adjuvant.
IM = Intramuscular.
ID = Intradermal.
criterion
statistical
dots
are volunteers
with the adjuvant. IM = Intramuscular. ID = Intradermal. The criterion for statistical significance
significance
was p <immunized
0.05.
was p < 0.05.
Gp41 epitope mapping
16
All 23 volunteers developed Env abs when samples were analyzed using whole gp160
protein. In order to determine the specificity of the abs, the samples were analyzed using
individual peptides from the gp41 protein. ELISA plates were coated with different peptides
with the length of 15 amino acids. Although the positive control samples (plasma from HIV-1
infected patients) worked in the assay, none of the volunteers had responses against any of the
peptides (data not shown).
17
DISCUSSION In this project I have investigated the humoral immune response in 23 healthy, Swedish
volunteers who were immunized with a genetic vaccine. The individuals were first primed
with DNA plasmids encoding HIV-1 antigens and thereafter boosted twice with a
recombinant viral vector, MVA, also encoding HIV-1 antigens. Already after the 1st MVA
boost, ab responses against Gag were observed in some individuals but no responses against
Env were detected [54]. This current project focused on the ab responses after the second
boost with MVA. One of the major aims of a HIV-1 vaccine is to induce B cells that produce
broadly Nabs. This has been shown to be very difficult as the most efficient abs are directed
only against a few conserved epitopes on the surface protein (Env) of the virus, and these
epitopes are in various ways shielded from access by abs. It is thus important that a vaccine
can induce Nabs against Env, but so far all vaccine-attempts have failed to achieve this [11,
58, 68]. Our vaccinees required two boosts of MVA to induce binding abs against Env and, as
compared to what is observed in infected individuals, the titers were moderate. The
presentation of HIV-1 is of course different in vaccinated and infected individuals and the
amount of antigen present after vaccination is much less than during an infection. Vaccinated
people receive HIV-1 DNA, which is transcribed and translated in our cells into proteins that
is in turn introduced to our immune cells. One of the major problems with developing a
vaccine against HIV-1 is the great variability of the virus. The viral enzyme RT lacks proofreading functions and this results in extremely high variation in Env amino acid sequence,
which in turn leads to escape from the immune system. However, the virus has the conserved
regions in the envelope but these epitopes are very well concealed from the immune system
by various mechanisms. The env protein is heavily glycosylated [66] and folds in a way
which conformationally masks the potentially neutralization sensitive epitopes
(conformational masking) [30]. This poses two problems, the first being how a vaccine
construct should be designed in order to be able to induce abs against these epitopes and,
secondly, even if you can induce the “right” ab with a vaccine the epitope on the real virus
will be masked.
Both Gag and RT abs cannot neutralize the HIV-1 virus, but we analyzed the Gag and RT
ab responses to analyze the immunogenicity of the vaccine. Gag-specific ab responses
increased after, and were detected, in all volunteers following the 2nd boost of MVA. A few
vaccinees also developed RT-specific ab responses, and interestingly, three of the vaccinees
already had abs in the baseline sample. As the RT is relatively conserved between different
retroviruses, this reactivity is most likely due to exposure to another retrovirus or from
retrotransposons found in our own genome [5]. The reactivity could, however, be boosted by
the HIV-1 vaccine.
Group specific responses did not relieve the best vaccination delivery method
The volunteers were vaccinated differently to investigate the optimal delivery strategy. Earlier
results have shown that volunteers immunized with DNA ID were able to induce similar
immune responses as those vaccinated IM. Addition of the adjuvant GM-CSF seemed to
reduce the responses when delivered ID together with the vaccine. It has also been shown that
the first MVA boost were more immunogenic when injected IM than ID [54]. Only 23
vaccinees received all immunization, resulting in too few individuals/group to make a proper
comparison between groups. Thus, no solid conclusion can be made regarding the optimal
route of delivery. However, the vaccinees in the group receiving both the DNA and MVA IM,
without GM-CSF, seem to respond slightly better than the other, although the difference is not
statistically significant.
18
Gp41 mapping
The gp160Env protein is divided into gp120 and gp41 after translation and both proteins
contain several conserved regions that is potential targets for Nabs. In order to determine to
which epitopes the abs in the vaccinees were directed, a peptide ELISA was performed using
peptides covering the entire gp41. However, no reactivity in any of the samples could be
detected against any of the peptides. The abs bound to the whole gp160 protein, but not to the
15 amino acid long sequences of the gp41 protein, possibly indicating that the majority of
responses are directed against conformational epitopes and cannot bind to the linear epitopes
represented by the peptides. We cannot be certain that this is the case but it is desirable to
induce responses against conformational epitopes as this indicates that the abs will bind the
native virus.
Future aspects
The volunteers will out of practical reasons not receive a 3rd MVA boost, but it would be
interesting to see if such a boost could increase the titers.
In the HIVIS-study, both needle injection and the Biojector system have been used when
delivering the vaccine. There will probably be a new trial set up further on using a different
and new approach including a new delivery method. A HIVIS program is also running in
Tanzania and this program includes 60 healthy uninfected volunteers. The program has been
successful when it comes to immunogenicity and safety and a new trial with a larger group of
volunteers will start during 2010 [2].
The experience of existing vaccines and extensive studies of the virus, suggests that an
effective vaccine will have to induce both humoral and cellular immunity. When designing a
vaccine that should elicit Nabs there are a few things that need to be considered: The abs seem
to be conformational sensitive and the immunogens should consequently not consist of
peptides. The immunogen should be constructed in a way that would induce abs against the
conserved, immunogenic regions of Env. Moreover, it would be beneficial to induce abs that
interfere with the required conformational changes in gp41 and thus prevent the fusion of
virus and cellular membrane. Possibly, it is better to concentrate the humoral response against
a few neutralizing epitopes rather than to use big molecules containing many epitopes.
In the wait for a vaccine, it is highly important that other preventive and therapeutic
means are implemented. For instance, condom use, male circumcision [45], access to
HAART, education, strengthening of women’s rights and the fight against stigmatisation of
HIV-1 infected persons should be the top priorities in developing countries. These are matters
that will be important for stopping the epidemic, even if a protective vaccine is developed.
19
ACKNOWLEDGEMENTS
I would like to thank Britta Wahren for giving me the opportunity to do this project at the
Institute and for her great encouragement and for introducing me to the fascinating virus.
Andreas Bråve, my supervisor, for his guidance and for the scientific discussions.
Jorma Hinkula, my co-supervisor, for his great support and scientific knowledgement.
David Hallengärd for all the discussions and laughs in the lab and in the office; we were
great in the movie!
Gunnel Engström for helping me to find lab materials, Maria Isiguliants for your positive
thinking and Susanne Johansson for all your help when analyzing results.
Anne, Karin, Nina, Christoffer, Caroline and Michaela for making the atmosphere at SMI
even better.
Andreas, my family and all my friends, I could not have done this work without your
support!!!
20
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25
Appendix 1
Table 2. Endpoint titers in volunteers at different time-points.
Volunteer
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Baseline
2 w post 1 MVA
40 w post 1 MVA
2 w post 2 MVA
(RT/Gag/Env)
(RT/Gag/Env)
(RT/Gag/Env)
(RT/Gag/Env)
(RT/Gag/Env)
(1/1/1)
(1/1/1)
(1/1/1)
(N/A)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(82/1/1)
(1/1/1)
(1/1/1)
(78/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(109/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/1142/1)
(1/1/1)
(1/1336/1)
(-/1402/1)
(1/1/1)
(1/1/1)
(1/1/1)
(1/527/1)
(1/1/1)
(1/690/1)
(1/1/1)
(93/1/1)
(1/6132/1)
(1/1/1)
(141/1/1)
(1/108/1)
(1/1774/1)
(1/1/1)
(1/73/1)
(120/121/1)
(1/1806/1)
(1/1805/1)
(1/1/1)
(1/1/1)
(-/966/1)
(-/1/1)
(-/425/1)
(-/1238/1)
(-/1/1)
(-/1/1)
(-/1/1)
(-/1324/1)
(-/1/1)
(-/2095/1)
(-/1/1)
(-/1/1)
(-/5188/1)
(-/1/1)
(-/1/1)
(-/51/1)
(-/980/1)
(-/71/1)
(-/54/1)
(-/112/1)
(-/1351/1)
(-/1350/1)
(-/1/1)
(-/1/1)
(-/1201/118)
(-/1976/388)
(-/8241/673)
(N/A)
(-/1843/1301)
(-/519/134)
(-/620/355)
(-/1666/424)
(-/600/485)
(-/3262/35)
(-/820/120)
(-/5667/204)
(-/2417/293)
N/A
(-/579/354)
(-/2991/207)
(-/2070/409)
(-/1952/1037)
(-/2998/1696)
(-/2825/1098)
(-/2243/652)
(-/2751/1148)
(-/244/22)
(-/446/380)
(61/1053/54)
(1/1233/409)
(839/8566/728)
(N/A)
(1/1371/1042)
(1/308/80)
(1/334/236)
(1/1365/192)
(1/413/275)
(1/3072/11)
(1/720/175)
(153/3171/277)
(1/2012/860)
(1/5624/1723)
(280/518/281)
(1/2485/123)
(1/1647/365)
(132/1869/900)
(1/3548/1698)
(384/2682/1266)
(123/1671/455)
(42/2416/688)
(104/165/36)
(111/268/275)
26
4 w post 2 MVA