Human Immunodeficiency virus 1 (HIV-1)

1. Introduction
1.1Human Immunodeficiency virus 1 (HIV-1)
1.1.1 HIV epidemic and methods of transmission

Acquired Immunodeficiency Syndrome (AIDS) is caused by an infectious agent known as Human Immunodeficiency Virus (HIV). Since 1980 when HIV was first discovered, nearly 25 million people have died from AIDS and nearly 34 million were living with HIV in 2009 [1]. Over the past 25 years it has overshadowed all other forms of immunodeficiency and is currently considered a severe global pandemic. There are currently two recognised types of HIV; HIV-type 1 and HIV-type 2 with type 1 being the main agent of AIDS worldwide[2]. HIV is a virus which belongs to the lentivirus genus of the retroviridae family [2]. This is an important factor in that infections with lentiviruses tend to have a chronic course of the disease with a long period of clinical latency, persistent viral replication and infection of the central nervous system [2]. HIV is transmitted through blood and mucosal tissue via sexual intercourse, needle sharing in drug use, blood transfusion and breast feeding. Today the main transmission method of HIV is the exchange of bodily fluids between partners during sexual intercourse. The transmission frequency of HIV is determined by the amount of infectious agent present in the particular bodily fluid in relation to how much contact the other individual has with that body fluid. The determination of whether the infection is established is mainly based on three factors, considered as the three points of the classic epidemiological triangle. These points include:
Characteristics of the infectious agent.
Host related factors; includes such as how susceptible the host is and how their body’s immune system responds.
Environmental factors such as social, cultural and political status also have a part in the process[2].
Today the pandemic is fuelled predominantly by heterosexual transmission, compared to homosexual transmission in the early 1980s. The last decade has seen the majority of new HIV infection cases being established in women rather than males [2, 3]. This implies that there is increasing discrepancy between infection rates of men and women. It is possible that gender inequalities associated with socio cultural norms play a role in that condom use is generally in the control of the male partner. This situation is particularly true of developing countries [3].Some early studies revealed that there is a two to fivefold- greater risk of infection from male to female transmission. There is a low incidence of infection taking place during any exposure below 1:100 during unprotected heterosexual intercourse [4]. For HIV transmission to occur there needs to be an entry point (i.e. unhealthy or damaged mucosa) in the vagina or anal canal because normally, a healthy mucosa is an effective barrier to transmission. Venerial diseases or lesions at these specific points would weaken them therefore increasing the chance of transmission [4] . Furthermore, during penetration female genitalia becomes inflamed due to small scale trauma. This is significant because activated neutrophils which migrate to these inflamed areas bind HIV-1 and can thereby transfer the virus to target cells [5]. The virions or infected cells that enter the uterus can infect CD4+ T cells and macrophages that reside within the endometrium by entering the cells by transcytosis [6 , 7] (process by which macromolecules are transported across a cell into the circulation0. In addition interleukin 8 which happens to be the most prominent cytokine found in the female genital tract can increase HIV replication in T cells and macrophages [8].
1.1.2 HIV: The virus
The general structure and genetic layout of an HIV particle can be seen in figure 1. Just like other retroviruses the core structural proteins (p24,p7,p6) and matrix (p17) are encoded for by the gag gene. The viral envelope glycoproteins gp120 and gp41 whose job it is to recognise cell surface receptors and fuse membranes are encoded for by the env gene. The pol gene encodes for enzymes that are crucial for viral replication. These enzymes are reverse transcriptase, integrase and protease. The protease enzyme cleaves large Gag and Pol protein precursors into their functional components. Reverse transcriptase is responsible for converting viral RNA into ssDNA, while the intergrase incorporates the viral DNA into the host cells chromosomal DNA [2].
The HIV life cycle can be summarised into six steps: Binding and entry ; uncoating; reverse transcription; provirus integration; virus protein synthesis and assembly and budding [2].HIV’s viral envelope plays an integral part concerning how the virus binds to the cell. The envelope is a trimeric complex that is made of two heterodimer proteins- gp120 and gp41 [2].In addition to its fusion, it is essential for virus recognition and entry into target cells.

1.1.3 HIV entry and replication cycle
The entry of HIV-1 into the host cell and subsequent HIV infection is mediated by the interaction of viral envelope glycoproteins and proteins gp120 and gp41 with the cellular receptor CD4 and a co-receptor which is normally CCR5 or CXCR4 [11 ]. This combination allows the virus to fuse with the host cell membrane and enter the cell. The glycoproteins are derived by proteolytic cleavage of a trimeric, glycosylated gp160 envelope glycoprotein precursor 2 and 46 [11 & 12].When the gp120 binds to the cellular receptors there is a change in the viral envelope conformation which leads to the exposure of gp41-a hairpin like glycoprotein. This exposure initiates the fusion between the HIV virus and its target cell via insertion of the glycoprotein into the cell wall [13 & 14]. With one end of the gp4 attached to the cell wall and the other to the virus, the virus is able to pull itself close enough to the target cell membrane allowing it to fuse with the cell in a manner in which the inner part of the virion, the viral core and its associated RNA as well as the pre-integration complex enter the cells cytoplasm [13 & 14].
It was concluded after a study in Uganda [15] that the probability of HIV-1 heterosexual transmission is dependent on the viral load, as results showed that transmission was rare for the individuals with HIV RNA plasma levels of less than 1,500 copies per ml. This study is in agreement with the theory that a reduction in plasma viral load would decrease the chances of transmission [15].
As previously mentioned the three main principal method of transmission are via blood, sexual intercourse and mother-to-child. The risk of transmission can potentially be explained by the relative concentration of HIV in various body fluids , and studies have suggested that this high virus load can be observed in the blood during acute HIV infection or the symptomatic period [15 &16].
1.1.4 Clinical features of the HIV-1 infection

Symptoms of the HIV virus when it first enters the patient’s system can usually be observed in the first four weeks [17]. Such patients are likely to present with flu-virus-like illness known as acute retroviral syndrome (ARS, whose symptoms include: swollen lymph nodes; high and low grade fever; nonpuritic muscular erythematous rash around the trunk; oral candidias and diarrhoea in some and headaches amongst others [ 2,18, 19, 20]. While the rash can be difficult to diagnose it is a valuable diagnostic sign in the diagnosis of HIV as its appearance can distinguish between primary HIV infection and other types of infection. The presence of a rash during diagnosis could possibly be a reflection of antigen: antibody complexes in the skin [19].The above mentioned symptoms can last up to three weeks, followed by an asymptomatic period which can last from months to years.
The HIV infection causes one’s immune system to become dysfunctional through two processes. The first process is that it causes the depletion of CD4+ T-cell causing immunodefiency, and the second is that HIV causes immune activation caused by an inflammatory response to HIV which leads to immunosuppression. It is believed that immune activation is caused by a homeostatic response to CD4+ T cell depletion and the inflammatory response to HIV infection that occurs [21]. Many studies cited by Stenger [21] have shown that there is a correlation between the CD4+ T-cell count and the clinical manifestations of HIV infection as well as the progression of the infection to AIDS. This CD4+ T-cell count is additionally used as a marker as to when one should begin treatment [21].
The mucosal environments such as that of the vagina, cervix and foreskin, there are specific CD4+ dendritic cells known as Langerhans cells. These specific dendritic cells can be found specifically within the superficial epithelial layers of the vagina and foreskin of men [22, 23]. According to some research, studies [22,23,24] have shown these cells to be the targets for HIV as they appear to express more chemokine receptor type-4 (CXCR4) than chemokine receptor type 5 (CCR5). HIV uses both CXCR4 and CCR5 as co-receptors in order to enter their target cells but there is a reason it is more attracted to one than the other. CCR5 has specific ligands it utilises which are RANTES, MIP-1 beta and MIP-1 alpha [25]. It was reported [25] that these ligands have the ability to suppress HIV-1 infection in vitro . Viruses that use CCR5 can be isolated in the early stage of infection.
CXCR4 is an alpha chemokine receptor specific for stromal- derived -factor-1 ( SDF-1) , which is a molecule that is endowed with potent chemotactic activity for lymphocytes [26 ]. This chemotactic activity is of particular interest to HIV because the virus uses these kinds of receptor to infect CD4+ T cells. CXCR4 is readily found in the female genital tract because during the natural implantation window or hormone replacement therapy, CXCR4 is up regulated in the endometrium [26, 27].
1.2 Vaccines
HIV prevention is now regarded as an umbrella term encompassing structural, behavioural and biomedical preventative strategies. These strategies include vaginal microbicides, oral pre-exposure prophylaxis, and implementation of male circumcision, highly active antiretroviral therapy, male and female condoms, and vaccine development [28]. The most common method of HIV prevention is that of condom use, which has been demonstrated to be efficacious by both vitro and epidemiological studies in preventing the passage of the virus [29, 8, 30]. The use of a diaphragm in woman is also useful because transmission occurs mainly by the virus entering through the cervical os into the uterus [31]. The authors of [31] also suggested that in the last few years a lot of attention has been on male circumcision. They found that studies between African and Asian countries where the risk factors of heterosexual transmission are similar have shown lower HIV transmission rates are associated with higher rates of male circumcision. The same studies also showed that uncircumcised men had a two-fold increase in the risk of contracting HIV per sexual act as compared to circumcised men [31, 15].It would appear therefore, that it has become accepted that male circumcision can partially protect against HIV. In contrast, there are rare cases in females were individuals do not contract HIV despite being exposed to the virus via genital fluids of infected partners. These individuals have antibodies in their vaginal fluids that appear to neutralize HIV and possibly block virus infection [32,33].The suggested theory is that Antibody- dependent cell-mediated cytotoxicity (ADCC) directed against HIV-infected cells in vaginal/cervical fluids can be associated with a reduction in levels of infectious HIV is an idea this project browses past [32].It is widely acknowledged by researchers [28]that the ultimate strategy in the eradication of HIV/AIDS would be to develop a fully efficacious vaccine. This is still a challenge to date due to lack of knowledge and understanding about the process by which HIV-1 evades antibody-mediated neutralisation [28].
The aim of any vaccine is to reduce and prevent infection in a given population [34]. As it stands, the biggest issue currently in the HIV vaccine world is preventing HIV infection, especially in resource limited countries. Currently, most HIV vaccine approaches are directed at inducing adaptive immune responses, including neutralizing antibodies and antigen-specific cellular immune responses. At the same time there is an appreciation for the need of an effective innate immune response [35, 36]. The need to evoke an innate response was triggered by a study in rhesus monkeys that were intravaginaly inoculated with Simian Immunodeficiency Virus (SIV) leading to a small focus of virus infection. It was concluded that if the virus replication at that site is suppressed, then the infection cannot be established as the early reaction of the innate cells is able to kill incoming virus infected cells at mucosal surfaces or within lymph nodes-or better yet suppress the virus preventing it from spreading [36, 37].
One of the biggest problems when it comes to vaccine development is finding an appropriate animal model of HIV infection. Generally the immunogenicity of a potential vaccine is first tested on animals like rabbits or mice where the immunogenicity is evaluated. If the results are desirable human trials of the vaccine will begin.
Microbicides currently have the spotlight in the vaccine world as they have the potential to become a good prevention for the disease. These are products; namely gels, that can be applied prior to sexual intercourse to prevent the transmission of HIV. Microbicides also known as Rheologically structured vehicle (RSV) gels were designed to be a delivery system for vaginal mucosal vaccinations [28]. Recently a double-blind, randomized control trial was conducted comparing tenofovir gel with a placebo gel in sexually active HIV-negative women in South Africa. The trial was to test the effectiveness and safety of the tenofovir gel, which is an Antiretroviral microbicide designed for the prevention of HIV infection in woman[38]. At the end of the study the tenofovir gel reduced HIV acquisition by up to 39%. There was however no changes in viral load and no tenofovir resistance in HIV sero-converters were detected. Therefore tenofovir gel could potentially fill an important HIV prevention gap [38].

1.2.1vaccine trials
Along with microbicides, today’s vaccine world there are several vaccines that are at the phase one and two stages. Many large trials have been conducted in Thailand, where several microbicide and intramuscular vaccines have reached phases one and two with some getting the go ahead for efficacy evaluation. The HIV epidemic in Thailand began in the 80’s; with the introduction of HIV-1 subtype B among injectable substance users followed by sexually transmitted subtype E [39]. Thailand’s first national plan for a vaccine development programme began in 1993 and since then they have been successful in controlling the heterosexual spread of HIV. The recombinant rgp120 vaccine was selected for evaluation on the basis of safety and immunogenicity profiles in humans. A phase 1 & 2 trial of a monovalent subtype B rgp120 vaccine among intravenous injection drug users in Bangkok was successfully conducted in 1995-1996, and was followed by a similar trial of a bivalent subtype B/E rgp120 vaccine in 1998 [39]. These trials were an important milestone as they proved that rgp120 was safe and immunogenic.
In 2004 a similar phase 1/2 safety and Immunogenicity of an HIV subtype B and E Prime-Boost Vaccine Combination in HIV-negative Thai adults was conducted using the candidate vaccines ALVAC-HIV (vCP1521) and AIDSVAX B/E. These two vaccines were developed based on the gp120 from macrophage tropic (r5) strains of CRF01_AE, in combination with antigens from subtype B HIV-1 [40]. The results of this trial showed that this vaccine combination of ALVAC-HIV (vCP1521) and either dose of AIDSVAX B/E were well tolerated and immunogenic. A dose response between AIDS-VAX B/E and antibody response was observed. The trial also demonstrated that the vaccines developed neutralizing antibodies to Thai E HIV and/or CD8 CTL responses to ALVAC-expressed HIV antigens. This trial reached milestones that allowed for advancement to phase 3 [40]. There was a phase 3 clinical trial of an HIV vaccine (VAX004) which was conducted in the US using a candidate HIV-1 gp120 vaccine, AIDSVAX B/B (VaxGen).The hypothesis from the US trial was that the antibodies directed against the envelope would bind ,neutralize and clear HIV particles before an infection could be established. The trial did not work as expected due to the lack of efficacy from VAX004 [41]. Subsequent vaccine trials have focused on eliciting cell responses. This is due to the presence of HIV-1-specific CD8+ T cells being correlated with the resolution of peak viremia during acute infection. The same trial found evidence of vaccine-specific CD8+ T cells in volunteers who received the vaccine regardless of behavioural risks. The CD8+ response was seen to be significantly high in participants who went on to contract HIV. This suggested that the AIDSVAX immunization may boost pre-existing immune responses-due to pre-infection exposure [41].
The future of HIV treatment is seeing a number of new experimental HIV drugs called Entry Inhibitors being designed to interfere with the interaction between CCR5 and HIV. One problem with this is that CCR5 is not the only co-receptor that can be used.
1.2.2 Vaccines: what is desirable

An effective immune response against HIV-1 would be one that is able to control and suppress viraemia during primary and chronic HIV infections. Simultaneously, you need something that would provide long lasting protection [42]. In the world of vaccines today, ideally it is essential for an HIV-1 vaccine to be able to elicit broadly cross-reactive neutralising antibody (Nab) responses against highly neutralization- sensitive strains of HIV-1 [43]. The antibodies will be required to be neutralizing ones as it has been recognised that neutralizing ones have the ability to employ multiple mechanisms that are able to interfere with viral replication [12]. As previously mentioned receptor binding and fusion is mediated by the envelope proteins and this happens to be an essential step in the life cycle and establishment of infection. This therefore makes it a fantastic target for neutralizing antibodies to have their effect by interfering with the fusion process and/or by neutralising free virions [12]. Given that most new infections of today are established in women as suggested before, it is within reason that there is a greater need to pursue and develop female-controlled preventative strategies. These will principally involve the cervix and vagina as the predominant mucosal portal of entry in heterosexual transmission, with the aim of eliciting sterilising immunity [28]. A multi-gene vaccine appears to be the best type of vaccine as it has been proven to have the potential to elicit broad, effective responses in animal models [42]. Understandably this kind of vaginal vaccine would need to be safe, cheap, easy to use, store and most importantly be able to induce long-lasting; high-titre protective mucosal and systemic response to diverse viral isolates through repeated and/or sustained female-controlled administration [28].
The last decade has shown that antiretroviral drugs considerably extend the lives of individuals infected with a virus, but a better solution for the epidemic would be the development of an effective and safe vaccine. So far in research, because the host immune system has not shown effective viral clearance of HIV, there is no model of protection that can be a definite emulate of a vaccine. The genomic diversity of the virus poses many barriers in the development of a good vaccine. It is therefore concluded that a good vaccine is one that would remove the virus before it can be established.
Recent studies have shown that B-cells can be stimulated to generate high titres of broadly cross-reactive neutralising antibodies against multiple genetic subtypes of the HIV virus [44]. Recent evidence [44] has suggested that some of these antibodies are directed against epitopes in the CD4 binding site on monomeric gp120, compared to many others that are directed against often neutralising epitopes. An increase in the knowledge of the molecular and antigenic structure of gp120 and gp41 HIV-1envelope glycoproteins (Env) has given new insights for vaccine design. However it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies [44, 45].
Based on the findings and evidence identified above, this project will attempt to look at vaccine response using a clade-C recombinant trimeric envelope glycoprotein CN54 gp140 as a part of the immunization response to elicit a humoral immune response. A broadly neutralising response is the desired outcome being attempted in this piece of work because the project ultimately aims to make the neutralising regions of the antigen more accessible. This is going to be achieved by immunization of rabbits to see if it elicits modified antibody responses on the envelope protein which will be fixed in different confirmations. If this is found to be true then a different immune response should be induced.

2. Materials and Methods
2.1 Immunogen
The recombinant trimeric envelope glycoprotein CN54gp140 was supplied by S.Jeffs (Imperial College, London). Intramuscular immunizations of CN54 gp140 was encoded by the CN54gp140REKR HIV-1 envelope gene cassette, derived from the clade-C/B’ HIV-1 molecular clone p97CN54 of Chinese origin developed by Wolf and Wagner, University of Regensburg, Germany.
2.2 Adjuvants
An adjuvant LASTS was added to the immunization to improve the immune response. The particular LASTS formulation used is an emulsion of MPLA which is monophosphoryl lipid A.
DS003 a small molecule known as BMS 599793 was added to two of the group immunogen formulas. It is an HIV entry inhibitor drug.
2.3 Rabbit immunization protocol/ In Vivo procedure-
24 Rabbits split into six groups were obtained and kept at St George’s University of London, London. All the procedures were performed in accordance with the Home office standards under the Animals Scientific Procedures Act, 1986, and approved by the Schools Ethical Review Committee.
Each group of rabbits was given four intramuscular immunizations at four week intervals containing 50µg of CN54gp140 in different formulations. A total volume of 1100µl was for each rabbit, 800µl was used per rabbit immunization. The formulations per rabbit and groups are as follows:
Group 1: 742.9 µl Wild type CN54 + 945µl LASTS+ 4612.1 µl PBS
2.4Immunization groups
A table showing the immunization groups that the rabbits belong to.
2.5Immunization and bleed schedule
A table showing the dates when Immunizations and bleeds took place.
Blood samples for serological analysis were taken before and after treatment according to the schedule above. Due to the death of several of the rabbits from undetermined causes, the final bleed and cull originally scheduled for 9th and 10th November 2010 was brought forward by four weeks to reduce the time frame for losing anymore rabbits. Blood samples were left to clot at room temperature for at least two hours. Blood samples were centrifuged at 4500rmp for 30 minutes and the sera was collected and re-centrifuged at 4500rmp for 10 minutes to remove any remaining red blood cells. The sera were recovered and stored at -80 degrees Celsius until needed.

The following reagents where used for the detection of IgG by ELISA: Phosphate buffered saline (PBS, 10X, BDH); Tween-20 (FISHER, Cat. No. BPE 337-500); Heat inactivated foetal bovine serum (FBS) (GIBCO, Cat. No. 10108-165); GMP HIV-1gp140 (POLYMUN); Mouse monoclonal anti-rabbit IgG (? chain – specific) HRP conjugate (SIGMA A1949); Sureblue TMB 1-Component Peroxidase Substrate (KPL, 52-00-02); TMB Stop Solution (KPL, Cat. No. , 50-85-06); Standard Rabbit antiserum to HIV-1 GB8 gp120 (NIBSC, Cat No ADP440. 1/R336); Positive control- Rabbit antiserum to HIV-1 GB8 gp120 (NIBSC Cat No: ADP440. 1/R546); Negative control- Normal rabbit serum (SIGMA, R9133)
2.6.1 Reagent preparation
1. Coating buffer, sterile PBS pH 7.4
1.1Prepare coating buffer by adding 50ml 10X PBS to 450ml de-ionised water.
2. Washing buffer, 0.1% TWEEN-20 in 1X PBS (PBST)
2.1Prepare washing buffer by adding 100ml 10X PBS to 900ml of deionised water. Add 500µ of TWEEN-20 and mixing thoroughly
3. Assay buffer, 10% FBS in PBST
3.1Prepare assay buffer by adding 10ml FBS to 90ml PBST
3.2Filter sterilise

2.7ELISA for the detection/quantification of HIV-1 gp140 IgG
An Indirect ELISA was decided as the best method to detect and quantify gp140 IgG in rabbit samples because; it as a specific assay and serum antibodies to HIV can be detected by this specific type of assay within six weeks of infection; in addition in this assay recombinant envelope and core proteins of HIV are absorbed as solid phase antigens to the wells. 96-Well plates (Greiner Bio-One medium binding) were coated with 50µl/well of HIV-1 gp140 at 5µg/ml in Phosphate Buffered Saline (PBS) for an hour at 37°C. The wells were washed (wash procedure was 4 washes in PBST) and blocked for one hour at 37°C with PBST with 10% sterile Foetal Bovine serum (PBST-serum). Standards, samples and controls were diluted in PBST-serum and incubated for 1 hour at 37°C. The wells were washed and bound antibody was detected using monoclonal anti-rabbit IgG (gamma-chain specific) Horseradish Peroxidase (HRP) conjugate (SigmaA1949) diluted 1:10 000 in PBST-serum and incubated for 1 hour at 37 degrees Celsius. After washing, the wells were incubated with 50µl TMB (Sureblue TMB 1-component peroxidase substrate (KPL) for five minutes in the dark. The reaction was terminated by the addition of 50µl of TMB stop solution (KPL) after five minutes incubation in the dark. The corrected mean of the quadruple absorbance (A450) measurements of each sample was obtained and compared with those of the negative controls on a microplate ELISA reader. A450 level is used because it produces the optimal results.
For the quantification of HIV-1 gp140 IgG, the first ELISA was used to screen all the serum samples. The endpoint titres of samples were obtained only when the absorbance measured at a wavelength of 450nm (OD450) was 0.2 or greater for samples diluted 1 in 100.Serial dilution of the samples were prepared in triplicates, and the reciprocal endpoint titres were calculated using SoftMax Pro GxP v5 software.

2.8Zeta Potential Measurements of gp140 constructs
The Zeta-potential, (?) of the gp140 molecules used for immunization was determined under a range of pH and salinity conditions with a Malvern ZetaSizer Nano ZS.
188 µl of the following gp140 constructs; WT, pH 4.0, pH 5.5 and pH 7.2 where all mixed individually into 10ml of 1X PBS. The sample was placed onto the zetasizer and a new cell inserted. Two titrants where also added in order to provide an acid and a base that could be added to the sample during titration in order to aid in the change of pH as the sample was titrated. The titrants were 1M HCl and 1M NaOH. The machine measures the zeta potential of the sample as the pH changes from 3 to 9, using every 0.5 interval as a target pH. The measurement for each sample was done in triplicates and each sample was measured three times. This protocol was repeated using deionised water and 154mM NaCl as the solution.

Data analysis was performed using GraphPad Prism; version 4.00 (GraphPad Software).One-way analysis of variance (one-way Anova) is a technic that is used to compare the means of numerical data. It requires a minimum of two samples to work. In this experiment, the one-way Anova analysis used Tukeys multiple comparison test to compare groups and immunizations, and data was considered statistically different if the p-value was less than 0.05. Furthermore, replicate data was assumed to be Gaussian distributed.

1. Results
3.1Zeta Potential Measurements
The Zeta potential of the gp140 molecules used for immunization was measured in triplicates under a range of pH and salinity conditions. The conditions were- 154mM NaCL, Deionised water and PBS. The averages of the zeta potentials where calculated and plotted against the average pH reached (Figures 3 and 4).
In PBS all the proteins show a strong correlation in response to change of pH. Statistically all the protein has a p-value that is less than 0.0001. The linear regression analysis shows that while the native protein has a slightly greater slope than the fixed protein there difference is not great. 1-way Anova analysis of the PBS data specifically at pH 7.5, where the graph indicates that the could be a difference, showed that there is no significant difference in the proteins. This test used Tukeys multiple comparison test, and a significant difference is considered to be when there is a p-value of less than 0.05.
In 154mM Sodium Chloride (NaCl) all the proteins have a p-value less than 0.0001in a correlation analysis, which again shows a strong correlation as a result of changing pH. A linear regression shows that the native protein has a significantly greater slope than the rest of the fixed proteins whose slopes have similar gradients. 1 way Anova analysis of the proteins in 154mM NaCl reveal that there is a significant difference with a p-value less than0.05 between the native protein and that fixed at pH 4.0. There is also a significant difference between the native protein and that fixed at pH 7.2. 1-way Anova analysis at a specific pH of 6.0 revealed several significant differences between protein groups. There significant differences noted were between: the proteins between fixed and 4.0 and 5.5; the protein fixed at pH 4.0 and 7.2; the native protein showed a significant difference when compared to all three proteins fixed at 4.0, 5.5 and 7.2.
In deionised water a strong correlation can again be observed in all proteins with a p-value of less than 0.0001 observed. Linear regression analysis reveals an extremely steep slope for the native protein compared to the other three fixed proteins. In addition native CN54 has the highest zeta potential measured in all the cumulative zeta potentials measures for each protein in any condition 1-way Anova analysis reveals that there is a significant difference between the native protein and that fixed at pH 5.5 and pH 7.2. 1-way Anova at the specific pH of 6.0 were it that from the graph the could be significant difference between the different proteins shows that there is a significant difference between all the groups when compared to each other. However there is no difference between the protein fixed at pH5.5 and one fixed at 7.2.
Looking at native CN54 in all three conditions, a steepest slope can be observed in the water condition followed by the 154mM NaCl and then PBS respectively. It is also noted that the native CN54 has a strong correlation in all three conditions with a p-value of less than 0.0001.1-way Anova analysis reveals a significant difference between the native protein in PBS and water, and also a significant difference between the native protein in water and 154mM NaCl. There is no difference between the protein in PBS and 154mM NaCl.
The protein fixed at pH 4.0 has the steepest slope (not as steep as that of the native protein) in water followed by PBS and then 154mM NaCl.1-way Anova analysis reveals a significant difference between the protein in PBS and in water , and a difference when in water compared to in NaCl. There is no difference between when the protein is in PBS and when it’s in NaCl.
When fixed at pH 5.5 the protein still has a strong correlation when measure in all three conditions with a p-value of less than0.0001. Once again the steepest slope can be observed in the water condition followed by NaCl and then PBS. The 1-way Anova analysis shows that no significant difference is detectable for the protein at pH 5.5 in all three conditions.
The protein when fixed at pH 7.2 shows strong correlation with a p-value of less than 0.0001. Regression analysis shows that the change is steepest in water followed by PBS and then NaCl. The 1way Anova analysis shows that there is a significant difference with a p-value less than 0.05 between the three conditions. There is a difference between the proteins when measure in PBS compared to in water. There is also a difference between the water and NaCl. There is however no difference between the PBS and NaCl.
It has been observed that adding an adjuvant to an immunization would improve the immune response. One potent adjuvant is the molecule Monophosphoryl Lipid A (MPLA) which is a component of bacterial cell walls, and has been used extensively in previous immunization studies because of the activation of dendritic cells through TLR4. There is also an inflammatory response, potentated through CD14 binding, which is dangerous to induce in the context of HIV, due to the increased susceptibility of infection. Modifications have therefore been made to the molecule to remove this effect, but still retain the adjuvanting properties, with the resulting formulation that is known as LASTS. This emulsion was added to each immunization. DSOO3 an entry inhibitor drug was added to a couple of the immunizations. The immunizations were administered over 12 weeks, and the end point titre of the HIV-1 gp140 IgG were tested in serum over time. No side effects were observed in the rabbits as a result of the immunization regime. However, during the immunisation schedules and different time points, a total of seven rabbits died randomly and the cause of death was inconclusive and unrelated to the immunizations.
All of the rabbits had a strong immune response against both the native antigen and the fixed antigen. The strong response in all the rabbits reached its peak at the second immunization and plateaued thereafter showing no significant increase in immune response between the second and final (fourth) immunization.
Groups four and five are the two groups that seem to have yielded some of the highest end point titres when titrated against both the native antigen and he fixed antigen. Overall group three rabbits which were immunised with the native antigen combined with the envelope stabilizing HIV entry inhibitor drug DS003 produced the highest titre when titrated against the native antigen. It is also potentially important to note that this group was comprised of only three rabbits and two of them died after the second immunization, making any conclusions which can be drawn weaker than those of higher numbered groups.
Group six which also had the HIV entry inhibitor drug DS003 combined with antigen fixed at pH 4.0 did not show a similar response. The peak immune response (129627.2) when measure against both the native and fixed antigen was reached after the second immunisation but it was not high as that observed in group 3 (338988.3). After the second immunisation in group three a further slight increase is observed (338988.3 to 712687.5) where as in group 6 a plateau is observed, with a possible slight decline (129627.2 to 57698.9)
3.2.4Grouped analysis of end point Titres
The immune responses for the rabbits all appeared to plateau without much increase after the second immunization. Figures seven and eight show the grouped end point titres after the second immunization, while figures nine and ten look at the grouped endpoint titres at the final bleed after the fourth and final immunization. The graphs show that there is a significant increase in the concentration of IgG from the second immunization compared to the final bleed. One-way Anova analysis of the second immunization titrations with the native and fixed antigen revealed that there was no significant difference between the groups. P values of the one-way analysis of variance were 0.3565 for the native and none was measured for the fixed antigen.
The most important results to consider are those at the final bleed between the native and fixed antigen. Group one and group three were only titre against the native antigen because they were not immunized with a fixed one. From figure 9 it is evident that these two groups produced high titres than those rabbits that were immunized with a fixed antigen. That is with the exception of group 5 whose antigen was fixed at pH 4.0. This group produced high end point titre results when the serum was measure against both the native and fixed antigen.
One way Anova analysis at the final bleed for both graphs in figure 10 against the native and fixed antigen revealed no significant difference in the groups.
4. Discussion
4.1 Results overview
This project aimed to characterise the physical and immunological properties of CN54 gp140 trimer and the effects that chemical fixation under different conditions confers. This study assesses the ability of intramuscular immunisation of rabbits with the vaccine trimer gp140 fixed in three different conformations and in combination with DS003 to elicit a modified antibody response, measured by the immunogen specific and native CN54 trimer specific end point serum titre.
Results show that the immunizations induced high serum CN54 gp140 specific IgG responses. Fixing the protein did not increase the humoral response above that observed with the wild type protein. The protein fixed at pH 5.5 was the only group to have an increased humoral response, but this did not reach statistical significance. Otherwise overall, the wild type protein induced a high immune response. The addition of DS003 to group 6 whose protein was fixed at pH 4.0 did not produce an increased response. However, addition of DS003 to group 3 whose protein is not fixed induced the highest immune response. The differences that can be detected between groups are limited due to the deaths of several of the rabbits from causes unrelated to the immunisation regime.
4.2 Fixed and unfixed protein
4.2.1 Zeta potential
The physical properties of the proteins were assessed by the changes in their zeta potential. Whether the protein is fixed or not has a profound effect on resulting surface chemistry. The paraformaldehyde will reduce the flexibility and crosslink the protein in the conformation that it exist in, reducing the variability in its higher order structure, and keeping it more similar to the conditions under which it was fixed.
Comparing the zeta potential in NaCl and water, the effect of fixation is it dampening the change in zeta potential while the samples where titrated. This is especially interesting because when the protein is titrated in PBS it is in a buffered system meaning there is a lot of different ions present causing the zeta potential to not be not well pronounced. The phosphate ions can act as a shielding factor, masking the alterations that fixation has induced. In NaCl however, you have a more fundamental system and other compounding factors that would be otherwise be present in the PBS have removed stripped and only sodium and chloride ions present. In water there are no other ions present providing a highly pure environment for the zeta potential measurements. As a result when the zeta potential of the native protein was measured in water and NaCl it is observed that there is a definite change in zeta potential as a result of pH. In all cases, the zeta potential becomes more negative as the environment becomes more alkaline. This fits with the theory that the zeta potential of proteins is made up of titrating the functional groups. At low pH conditions, there is an abundance of hydrogen ions which will confer a positive charge on protein in solution. The opposite is true when the system becomes basic and the system is dominated by hydroxide ions. The difference in how the proteins react differently to the changing conditions is represented by the steepness of the native protein slope on the graphs. This zeta potential change cannot be accounted for in the PBS buffered system. While there is a change it is not a pronounced one and the native protein behaves the same as the fixed ones. A change in zeta potential as a result of change in pH is also present in the fixed protein in the water and NaCl system, but the changes are not pronounced. The fixed proteins do not show as much of a change in the PBS system just like the native protein but it is concluded that this is due to the buffered environment that is provided by the PBS. In water and NaCl there is much more of a change but not to the same level as the native protein. This lack of change can likely be explained by the process of fixation of the protein. Because these proteins are so strongly correlated to changes in pH it is therefore hypothesized that fixation in those pH conditions would preserve the changes. Fixation of the protein is accomplished using paraformaldehyde. As a result the formaldehyde reduces the protein’s flexibility and crosslinks the protein in the conformation that it exists in. This means that the protein will have reduced variability in its higher order structure.
4.2.2End point titres
Figures 5 to 8 show that all of the rabbits had a strong HIV-1 gp140-specific IgG immune response against both the native and fixed antigen. This response appears to plateau after the second immunization and holds steady up to the final immunization. This means that after the second immunization subsequent immunizations are not inducing any more of an immune stimulation -they are just boosting what is already there. One could ask what is the relevance of this result in terms of vaccine development and if you only need to administer two vaccines. The plateau does not necessarily mean that only two immunizations are required, and this experiment cannot fully answer that. To determine the complete answer, a study would need to be conducted where only two immunizations are administered and the subjects are monitored over time without giving any more immunizations to see if the immune response lasts and for how long. This would be important because ultimately for a vaccine to be considered good you would want it to produce a response that is protective and lasts. You would also aim for a vaccine that gives you the biggest response using as little immunogen as possible.

4.3 Addition of DS003
DS003 is a small molecule also known as BMS 599793 and it was added to the immunogens of group 3 and group 6-with group 3 being a being made up of the wild type protein and the group 6 protein are fixed at pH 4.0. DS003 is an HIV entry inhibitor drug that blocks entry of the HIV virus by interacting with gp120. Currently it is being developed as a mirobicide which is proving to be very potent [46].The interest in using it in this study though is due to its reported effects on decreasing the flexibility of gp140 molecules. Stabilization of the protein through fixation may be additive to the effect of DS003.
As mentioned above, in order for infection to occur the viral envelope protein must bind to the CD4 receptor of the target cell. This binding occurs by the gp120 glycoprotein. A compound like DS003 would abrogate this process and encouragingly, it has been shown to prevent infection in vitro. Earlier studies have shown that DS003 was chosen specifically because it can bind to CN54 and gp140 trimers as well as gp120 monomer-and all these three envelope constructs bind soluble CD4. This is good because DS003 binds to the CD4 binding site on gp140.The benefit of such entry inhibitors is that they act early in the early stages of the virus cycle before infection can be established [46, 47].
Figures 7 and 8 show serum titration results for rabbits that were immunized with an immunogen that was combined with DS003. Both groups had a strong immune response to the immunogen reaching a peak after the second immunization and eventually plateauing. Group 6 however did not produce titres as high as group 3. The maximum titre produced in group 6 measured after the second immunization was 129627.2 and subsequently decreased by the final immunization, whereas the highest titre measured in group 3 was observed after the final immunization and was measured at 712687.5. This difference could likely be as a direct effect of the protein being fixed in group 6. This could mean that DS003 is not as potent when combined with a fixed protein. It shows that fixing the protein reduces the magnitude of effect that is stimulated in the immune system. This is supported by the fact that when combined with the native protein DS003 is able to exert its effects to the maximum. Seeing as the addition of DS003 to a wild type protein produced such high titre results, it could mean that the titre results of group 1 which were immunized with wild type protein alone could be potentially higher if DS003 were to be added, as seen in group 3.
With that in mind group 1 produced higher titres especially after the second immunization and the final time point than some of the rabbits that were fixed at pH 4.0 and 7.2. These differences were small though, and not statistically significant.
Overall looking at figures 9 and 10 groups 1, 3 and 5 produced the highest immune responses that were measured by titration. Group one only has the native protein; group three is a combination of the native protein with DS003 and group 5 is the only one with a fixed protein at pH 5.5. This could be interpreted to mean that with respect to the results from groups 1 and 3, the best immune response is produced when a native protein is used as an immunogen. DS003 increases the immune response best when it is in combination with a wild type protein rather than a fixed one as illustrated in figures 7 and 8. This could be due to fixation removing the binding site for DS003, and therefore leaving no opportunity for it to work. Group five shows that if an immunogen is going to be made from a fixed protein the best pH is 5.5. An experiment that could be conducted is combining DS003 with a protein fixed at pH 5.5 to see if it would have an impact on the immune response produced. This was not possible in the current experiment due to a limitation in the number of animals available.
4.4 Native and wild type protein.
An imperative factor in the race to design inhibitors and vaccines for HIV is to gain a good understanding of the different conformational states available to the HIV-1 envelope glycoproteins [11].The CN54gp140 immunogen that was used in the rabbit immunizations was successful in eliciting a strong, specific humoral antibody response. The immunogens used in these kinds of experiments are usually protein based ones adapted to mimic HIV envelope proteins on a whole virion. Using protein based vaccines can be difficult because the proteins have a complex structure and are usually fragile [28]. CN54gp140 manufactured under GMP conditions is very comparable to the wild type protein, and is used in this study because it has been shown to be exceptionally stable in buffered solutions. This protein was manufactured to mimic the actions of the native protein gp120 and gp41 molecules [28]. Because the wild type protein was so stable it was possible to fix the material easily in different conformations that were able to elicit a good immune response. Gp120, as previously described, works in conjunction with gp41 to allow the virus to get close enough to the cell membrane and inject its genome into the target cell cytoplasm. The difference between the native gp140 and wild type infectious protein is that the gp140 is not near the target cell membrane. There is also a mutation on the cleavage site that would normally result in a gp41 and gp120 molecule being created instead of a single gp140-but the same external face and glycosylation patterns on both types of proteins will be the same.
4.5 Conclusion
This study was successful in revealing whether vaccine response using the trimer CN54gp140 fixed in different conformations would elicit a modified antibody response. A decreased titre of antibody concentration was observed when the protein was fixed at pH 4.0 and 7.2 and there was only an increase in serum antibody in the protein fixed at pH 5.5. However the best response was seen in the immunizations with the wild type protein, especially in the group three where the protein was combined with the HIV inhibitory drug DS003.
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