Volume 4, issue 2 | FeBRuARY, 2013 CLOSING IN ONVolume 4, issue 2 | FeBRuARY, 2013 a Publication of...

Volume 4, issue 2 | FeBRuARY, 2013

a Publication of the HiV Vaccine Trials Network

immunogenicity Data Patterns emerging from Cross Trial Comparisons

Can We Be more effective in Designing Pre-efficacy Vaccine Trials?

2200 strong: Celebrating our Volunteers

Broadly Neutralizing monoclonal Antibodies: Powerful Tools for HiV Vaccine Development

seattle HVTu Helps Pilot HANC Native American initiative

sPeCiAl ANNouNCemeNTs [back cover]

CAleNDAR [back cover]

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A major advantage for vaccine studies conducted by the HIV Vaccine Trials Network (HVTN) is the uniformity with which the trials are conducted and evaluated. This includes maintaining similar key study design elements, as well as standardized methods for operational procedures, clinic procedures, data capture, and immunogenicity assessments. This consistency facilitates efficient trial implementation and ensures robust immunogenicity results.1 It also provides the ability to compare immunogenicity data across mul-tiple trials.

Enabling such comparability for trials conducted worldwide was a pioneering goal for the Network

when it was created in 1999. Since then over 60 trials have been initiated, evaluating diverse vaccine plat-forms from a variety of product developers. Here, we describe several informative patterns in immunogenic-ity that have emerged from cross trial data compari-sons.

Heterologous vector prime/boost regimens elicit higher T-cell response rates, while homologous regimens elicit higher antibody response rates

Prime/boost regimens administer multiple vaccina-tions as a means to increase immune responses to a

Immunogenicity Data Patterns Emerging from Cross Trial ComparisonsTracey Day, Barbara Metch, Nicole Frahm, and Cecilia Morgan

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2 FEBRUARY 2013 Volume 4:2 | HVTNEWS

immuNogeNiCiTY DATA PATTeRNs emeRgiNg FRom CRoss TRiAl ComPARisoNs

vaccine. Homologous vector prime/boost regimens com-prise administrations of the same vaccine vector, whereas heterologous vector prime/boost regimens contain differ-ent vaccine products.

Several HVTN clinical trials (HVTN 055, 065, and 068) have evaluated whether homologous or heterolo-gous vector prime/boost regimens are better for different types of HIV vaccine platforms. By compiling the posi-tive response rate data from these trials, a similar pattern was observed (see Table 1 for trial descriptions).2-4 In each study, whether comparing pox virus vector combi-nations, or DNA plus modified vaccinia Ankara (MVA) or recombinant adenovirus serotype 5 (rAd5) vectors, the

heterologous boost regimen arms elicited higher HIV-specific T-cell response rates, and the homologous regi-men arms elicited higher HIV-specific antibody response rates. An example of this from HVTN 055, which compared MVA and fowlpox vectored vaccines, is shown in Figure 1. This trend was apparent despite the use of different vaccine vectors and inserts, suggesting that this pattern could hold true for other vaccine types.

Both antibody and T-cell responses may be important for a successful HIV vaccine. How might we get the best of both worlds with prime/boost regimens? This is not yet known, but a clue may come from results from HVTN 078. This study compared different heterologous

Figure 1. Heterologous vector prime/boost regimens elicit higher T-cell response rates, while higher binding antibody response rates are induced for ho-mologous regimens. Relative peak immunogenicity response rates for T-cell (blue) and antibody responses (red) for HVTN 055 (mean response rate with 95% confidence intervals). M refers to MVA vector and F to fowlpox vector. The response rate data from the homologous arm with FPV-HIV were omitted due to low immunogenicity of this regimen.

STaNdardIzEd prOCEdUrES USEd by THE HVTN CLINICaL SITES

include isolation and cryopreservation of plasma, serum, and peripheral blood mononuclear cells (pbMCs). Study specimens are shipped to HVTN endpoint laboratories where assays analyzing T-cell and antibody responses are performed. For T-cell responses, validated assays include antigen-specific cytokine expression via intracellular cytokine staining and IFN-γ ELISpot.16-19 For antibody responses, standardized assays include binding antibody multiplex assay or ELISa for binding antibodies, and the TzM-bl and a3r5 assays for neutralizing antibodies.19,20

The HVTN Statistical and data Management Center (SdMC) and the HVTN Laboratory program have developed statistical methods to determine whether a participant has a positive response in an assay. The percentage of participants with a positive response (ie, positive response rate) is the value often used to compare immunogenicity across trials, as well as between individual arms within a trial.

HVTNEWS | Volume 4:2 FEBRUARY 2013 3 www.hvtn.org


ProTocol ProducTs BY acTive arms

collaBoraTors Hiv GeNe iNserTs / ProTeiNs

sTudY FiNdiNGs* (comPleTed Trials)/sTudY aims (oNGoiNG aNd PlaNNed Trials)

HVTN 044 4-plasmid dNa; IL-2/Ig dNa; 4-plasmid dNa + IL-2/Ig dNa t0; 4-plasmid dNa + IL-2/Ig dNa t48

VrC, daIdS gag, pol, nef, env plasmid IL-2/Ig significantly increased immune responses when administered 2 days after the dNa vaccine, compared with simultaneous administration.

HVTN 052 4-plasmid dNa VrC, daIdS gag, pol, nef, env appeared safe and well tolerated. Three doses of the 4-plasmid dNa product elicited more T-cell responses (50%) than 2 doses (22%), primarily a Cd4+ T-cell response.

HVTN 055 rFpV; rMVa; rMVa + rFpV

Therion, daIdS env, gag, tat, rev, nef, RT The MVa-HIV and FpV-HIV vaccines were well tolerated, and heterologous boosting was superior at inducing Cd8+ T-cell responses.

HVTN 060 dNa gag; dNa gag + IL-12 dNa; dNa gag + IL-12 dNa + CTL MEp

Wyeth (pfizer), daIdS gag The HIV gag dNa with plasmid cytokine adjuvants was well tolerated. There were minimal responses with HIV gag dNa alone and a non-significant increase in response rates only with the intermediate dose of IL-12.

HVTN 063 dNa gag; dNa gag + IL-15 dNa; dNa gag +IL-15 dNa + IL-12 dNa

Wyeth (pfizer), daIdS gag The HIV gag dNa with plasmid cytokine adjuvants was well tolerated. There were minimal responses with HIV gag dNa alone, and no apparent augmentation with IL-12 or IL-15.

HVTN 065 rMVa; dNa + rMVa GeoVax, daIdS gag, pr, RT, env, tat, rev, vpu, pol

MVa/HIV62 elicited different patterns of T-cell and antibody responses when administered alone (higher antibody responses) or in combination with the JS7 dNa vaccine (better T-cell responses).

HVTN 068 rad5; 4-plasmid dNa + rad5

VrC, daIdS gag, pol, nef, env Homologous rad5 boosting enhanced Env-specific antibody responses, but did not increase HIV-specific T-cell responses. despite limited post prime immune responses, dNa priming enhanced the magnitude of post boost Env-specific antibody and Cd4+ T-cell responses and influenced the cytokine and memory marker profiles of the boosted T-cell responses.

HVTN 070 dNa; dNa + IL-15 dNa; dNa + IL-12 dNa

U penn, Wyeth (pfizer), daIdS

env, gag, pol Together with HVTN 080 results, indicates electroporation with cytokine adjuvant increased responses to dNa vaccination significantly.

HVTN 071 rad5 Merck, daIdS gag, pol, nef Last study conducted with Merk rad5 vaccine, specimens from large blood draws and leukaphoresis have been used in well over a dozen ancillary studies to help understand the Step study results.

HVTN 076 dNa + rad5 VrC, daIdS gag, pol, nef, env an ongoing phase 1b clinical trial to evaluate mucosal immune responses following intramus-cular injections of an HIV dNa plasmid vaccine prime, followed by an HIV adenoviral vector boost in healthy, ad5 seronegative HIV-1 uninfected adults.

HVTN 077 rad35 + rad5; dNa + rad5; dNa + rad35

VrC, daIdS env an ongoing phase 1b trial to evaluate the safety and immunogenicity of recombinant adeno-viral serotype 35 (rad35) and serotype 5 (rad5) HIV-1 vaccines when given as a heterologous prime-boost regimen, or as boosts to a recombinant dNa vaccine in healthy, ad5-naïve and ad5-exposed, low risk, HIV-1 uninfected adult participants.

HVTN 078 NyVaC + rad5; rad5 + NyVaC

VrC, EuroVacc, daIdS env, gag, pol, nef an ongoing phase 1b clinical trial to evaluate the safety and immunogenicity of heterologous prime/boost vaccine regimens (NyVaC-b/rad5 versus rad5/NyVaC-b) in healthy, HIV-1 uninfected, ad5 neutralizing antibody seronegative adult participants.

HVTN 080 dNa; dNa + IL-12 dNa VGX (Inovio), profectus, daIdS

env, gag, pol Together with HVTN 070 results, indicates electroporation with cytokine adjuvant increased responses to dNa vaccination significantly.

HVTN 087 dNa + rVSV; dNa + IL-12 dNa + rVSV

profectus, daIdS gag, pol, nef, tat, vif, env an ongoing trial to evaluate the safety and tolerability of a prime-boost regimen of HIV-MaG vaccine given with and without plasmid human IL-12, delivered with electroporation, followed by VSV HIV gag boost in healthy HIV-1 uninfected adult volunteers.

HVTN 088 rgp140 with MF59 Novartis, daIdS Env an ongoing phase 1 trial to: 1) Evaluate the safety and tolerability of and 2) Evaluate neutraliz-ing antibody responses to HIV-1 Sub C gp140 vaccine with MF59 in healthy, HIV-1 uninfected adults, primed or unprimed with HIV-1 subtype b envelope subunit vaccines with MF59.

HVTN 094 dNa with GM-CSF + rMVa

GeoVax, daIdS pr, RT, tat, rev, vpu, env, gag, pol

an ongoing trial to evaluate the safety and immunogenicity of a heterologous prime-boost vaccine regimen of GEO-d03 dNa and MVa/HIV62b vaccines in healthy, HIV-1 uninfected vaccinia naïve adult participants.

HVTN 097 aLVaC + rgp120 Sanofi pasteur, Global Solutions for Infectious diseases, daIdS

env, gag, pr; Env a planned phase 1b clinical trial to evaluate the safety and immunogenicity of the vaccine regimen aLVaC-HIV (vCp1521) followed by aIdSVaX® b/E in healthy, HIV-1 uninfected adult participants in South africa.

HVTN 098 dNa; dNa + IL-12 dNa Inovio, daIdS env A, env C, gag, pol a planned trial to evaluate the safety and tolerability of pENNVaX®-Gp with plasmid IL-12, given by intradermal or intramuscular injection with electroporation, in healthy HIV-1 unin-fected adult volunteers.

HVTN 204 dNa + rad5 VrC, daIdS gag, pol, nef, env The regimen was well-tolerated and immunogenic, inducing HIV-specific IFN-ɣ+ T cells in 70.8%, which were balanced Cd4+/Cd8+ polyfunctional responses and high frequencies (83.7%–94.6%) of multi-clade anti-Env binding antibodies.

HVTN 205/908 rMVa; dNa + rMVa GeoVax, daIdS env, pol, gag, tat, rev, vpu, pr, RT

an ongoing phase 2 trial to evaluate the safety and immunogenicity of a prime-boost regi-men of pGa2/JS7 dNa and MVa/HIV62, or MVa/HIV62 alone in healthy, HIV-1 uninfected, vaccinia-naïve individuals.

* Text adapted from published manuscript.

abbreviations: VrC, Vaccine research Center (NIaId, NIH); daIdS, division of aquired Immune deficiency Syndrome (NIaId, NIH); rMVa, recombinant modified vaccinia ankara; rFpV, recombinant fowlpox virus; rVSV, recombinant vesicular stomatitis virus; rad5, recombinant adenovirus serotype 5; NyVaC, a highly attenuated vaccinia virus strain; GM-CSF, granulocyte-macrophage colony-stimu-lating factor; aLVaC, live attenuated recombinant canarypox derived virus; rgp120 / rgp140, recombinant glycoprotein 120/140 subunit; Ig, immunoglobulin; IL, interleukin

Table 1. Descriptions of HVTN trials mentioned in this article.



regimens with rAd5 and NYVAC vectors. The results of this phase 1b study found that priming with rAd5 and boosting with NYVAC was better than the reverse order.5,6 Importantly, this was true for both T-cell and B-cell responses, with both yielding promising response rates. Further research is needed, but these results suggest the hypothesis that employing a strongly immunogenic prime, such as rAd5, in combination with an immuno-genic boost may enable the best of both worlds by elicit-ing balanced T-cell and antibody responses.

In addition to heterologous and homologous vector regi-mens, the HVTN is investigating different insert regi-mens. HVTN 083, for example is an ongoing study that will examine the effects of heterologous versus homolo-gous inserts on T-cell response breadth.

enhancing responses with adjuvants and electroporation

DNA plasmids can be weak immunogens on their own, but several HVTN clinical trials point toward potentially effective ways to enhance DNA vaccine potency. One strategy yielding incremental immunogenicity increases has been the administra-tion of DNA vaccines with plasmid-encoded cytokine adjuvants. For example, HVTN 044 evaluated the effects of the cytokine IL-2 on the immunogenicity of an HIV plasmid DNA vaccine. In the study, IL-2 was deliv-ered as a plasmid-encoded fusion protein (IL-2/Ig) and given either simultaneously or 48 hours after the HIV DNA vaccine. Inclusion of IL-2/Ig resulted in a trend toward increased response rates by intracellular cyto-kine staining (ICS) when given 48 hours after the DNA vaccine.7 HIV-specific

CD3+ T-cell response rates measured by ICS 2 weeks after the final vaccination were 60% for DNA vaccine with plasmid IL-2/Ig given 48 hours later as compared to 44% and 30% for DNA vaccine alone and with simulta-neous IL-2/Ig administration, respectively (see Figure 2).

HVTN 060 and 063 evaluated the effect of plasmid cytokine adjuvants (IL-12 and IL-15) on the immuno-genicity of a truncated HIV-1 gag (p37) DNA vaccine. Though T-cell responses in general were low and there was not a statistically significant difference in response rates, there was an increase in HIV-specific T-cell re-sponses after 3 vaccinations when IL-12 was co-admin-istered at an intermediate dose, compared to DNA alone. T-cell responses were not observed at low and high doses of IL-12 and IL-15.8

More recently, potential benefits of using adjuvants in conjunction with electroporation (EP) as a delivery method for DNA vaccines have been observed. EP involves delivery of short electrical pulses after DNA vaccine injection. These pulses serve to increase DNA uptake by cells. In HVTN 080, 3 vaccinations of PEN-

Figure 2. Cytokine adjuvants and electroporation increase DNA vaccine response rates. The differences in CD3+ T-cell response rates between DNA vaccine alone and DNA given with a cytokine adjuvant (HVTN 044, 060) or elec-troporation plus a cytokine adjuvant (HVTN 070, 080) are shown. Note: not all study arms are shown (see Table 1 for study descriptions).



NVAXTM-B (3 mg dose) delivered via EP elicited a 39% higher positive ICS response rate compared to 3 vac-cinations of PENNVAXTM-B (6 mg dose) administered by standard intramuscular injection in HVTN 070. In addition, the use of adjuvants in conjunction with EP appears to be associated with further increase in vaccine potency. For example, in HVTN 080, 3 vaccinations of DNA with GENEVAXTM IL-12 delivered EP elicited a 61% higher positive ICS assay response rate compared to 3 standard intramuscular injections of the DNA vaccine alone (see Figure 2).9

Ongoing and planned HVTN studies will continue to investigate strategies to enhance vaccine potency via adjuvants or modes of administration. HVTN 087 is an ongoing study evaluating a DNA and vesicular stomatitis virus (VSV) vector prime/boost regimen in which the DNA will be co-administered with IL-12 and an EP device from another developer (see Table 1). A study cur-rently in development, HVTN 098, will compare intra-muscular administration of DNA via EP, with or without IL-12, with intradermal delivery via EP.

New cytokine adjuvants will also be investigated. For example, HVTN 094 is evaluating the cytokine granulo-cyte-macrophage colony-stimulating factor (GM-CSF) co-expressed with HIV genes on a DNA plasmid. Future studies may also evaluate various adjuvants with proteins for their ability to enhance immunogenicity and increase response durability of protein immunogens.

vector and insert enhancements to optimize platforms

Many vector and insert design improvements occur through an iterative process, with early phase HVTN trials and cross trial comparisons influencing preclinical and further clinical development. One example is the VRC DNA platform. The early generation product test-ed in HVTN trials consisted of a mixture of 4 plasmids: 1 with an env clade A gene insert, 1 with an env clade B, 1 with an env clade C, and 1 with a gag-pol-nef fusion gene. Results from HVTN 052 indicated Env-specific T-cell responses of approximately 30%, but responses to the other inserts was limited with 0% to Gag, 0% to Nef,

and 3.4% to Pol.10 The current VRC DNA construct is a mixture of 6 plasmids with each HIV gene insert on a separate plasmid. HVTN 204 demonstrated that sepa-rating the inserts onto individual plasmids maintained a T-cell response to Env (48.3%), while increasing the responses to Gag (48.3%) and Nef (27.6%).11

Another example of clinical trial results informing and improving next generation products comes from a com-parison of immune responses to different DNA vaccines in HVTN 080 and HVTN 204. Although overall HIV-specific T-cell response rates to the DNA vaccines were a little higher in HVTN 080, most of these responses were to Gag, and less to Env. In contrast, responses elicited by the DNA prime in HVTN 204 were more balanced against Env and Gag, as described above. These distinct response profiles identified opportunities to further improve vector and insert designs. The HVTN 080 developers indicate that preclinical evaluations of their next generation DNA vaccine, currently in development, demonstrate increased Env responses.

cross protocol analysis of HvTN dNa vaccine trials

To date, the HVTN has conducted numerous trials evaluating DNA vaccine products. Jin, Morgan, and Yu et al. compiled data from 10 HVTN DNA vaccine trials that used a validated ICS assay to investigate factors influencing DNA vaccine immunogenicity.12 Postvacci-nation responses from DNA only vaccine treatment arms (no adjuvants or EP) were compared.

Overall, the response rates varied considerably from study to study, but in all cases CD4+ T-cell responses occurred more frequently than CD8+ T-cell responses. The analysis showed that 3 administrations of a DNA vaccine elicited more frequent HIV-specific CD8+ T-cell responses than 2 administrations, and that a 4th admin-istration did not have a statistically significant effect on response rates in the regimens examined. In addition, there appeared to be a dose response up to 3 mg per vac-cine, but higher doses did not further enhance responses. These findings reveal trends across many DNA stud-ies and suggest potential ways to optimize this vaccine



platform.

The large number of participants included in the pooled DNA vaccine cross protocol analysis provided an op-portunity to examine potential associations between participant demographics and vaccine-elicited immune responses. Identifying host factors that influence vaccine immunogenicity can be useful for vaccine development in a variety of ways. For example, the information could be used to increase statistical power for assessing vaccine efficacy, and to improve the ability to assess immune cor-relates of protection.

Using multivariable logistic regression modeling, the cross protocol DNA vaccine study investigators evalu-ated whether participants’ age, gender, and body mass index (BMI) were associated with higher CD4+ and CD8+ T-cell response rates in the DNA vaccine trials. In this study, female gender and lower BMI were associated (independently) with statistically higher HIV-specific CD4+ T-cell response rates than male gender and higher BMI. A complimentary analysis was performed focus-ing on trials evaluating VRC DNA vaccines. Results from this analysis were similar to the broader analysis, as shown in Table 2. Similar trends have been noted in other studies.13 These results point to baseline character-istics that should be considered when evaluating vaccines in human populations.

innate immune responses predict T-cell response magnitude

Innate immune responses are another study readout that may enable predictions of candidate vaccine immune responses.14 Innate immune responses to pathogens are involved in triggering and shaping the ensuing patho-gen-specific adaptive response. Similarly, it is thought that innate responses to immunogens impact the char-acteristics of the adaptive responses that develop upon vaccination. The HVTN is working to define innate responses to vaccines that predict beneficial adaptive re-sponses. HVTN 071 was a phase 1b trial in which innate responses were evaluated by measuring gene expression profiles from PBMC over the course of 1 week following rAd5 vectored HIV vaccination.15 Differences in gene expression in PBMC were observed early after vaccina-tion, peaking at 24 hours postvaccination. Increased expression was observed primarily in genes involved in inflammation and interferon pathways, whereas expres-sion of genes involved in lymphocyte trafficking was decreased. Using a systems biology approach, innate gene expression profiles were compared with adaptive response characteristics. This method identified patterns in which levels of certain cytokine gene pairs and inflam-matory genes were correlated with HIV-specific CD8+ T-cell response magnitude. These results suggest that certain innate responses to the rAd5 vector HIV vaccine were predictive of T-cell response magnitude.

cd4+ (N=288 vacciNeesa) cd8+ (N=309 vacciNeesb)reFereNce GrouP esT. odds raTioc

(95% ci)Wald P-value esT. odds raTioc

(95% ci)Wald P-value

Female Male 2.22 (1.30, 3.80) 0.004 1.02 (0.48, 2.13) 0.96

body mass index 1 unit change 0.90 (0.85, 0.96) 0.001 0.96 (0.89, 1.04) 0.34

age 1 year change 0.99 (0.96, 1.02) 0.39 1.01 (0.97, 1.05) 0.55

a) 1 participant was excluded from the CD4+ model due to missing data for BMI and 23 due to lack of evaluable CD4+ T-cell assay data.b) 2 participants were excluded from the CD8+ model due to missing data for BMI and 1 due to lack of evaluable CD8+ T-cell assay data.c) The estimated odds ratios were adjusted for other variables in the model. The models also adjusted for race and number of plasmids in the vaccine, which were non-significant.

Table 2. Increased Env-specific CD4+ T-cell responses were associated with female gender and lower BMI in multivariable logistic regression models of CD4+ and CD8+ envelope specific T-cell response rates in 5 studies of the NIAID VRC DNA vaccine (N=312 vaccinees, 295 US and 17 Peru).



The HVTN Laboratory Program has developed a suite of innate response assays that may be used to assess in-nate immune responses in HVTN trials. Sampling time points allowing for these assessments have been incor-porated in multiple ongoing and planned trials evaluat-ing different products and regimens including HVTN 071, 076, 087, 088, 094, 097, and 205/908 (see Table 1 for trial descriptions). These assays will likely serve as a future platform for comparing cross trial data, and may help further identify innate responses that shape adaptive responses and potentially indicate induction of mucosal responses.

conclusion

Immunogenicity patterns emerging from cross protocol analyses of HVTN trials are providing clues for optimiz-ing candidate vaccine platforms and regimens. These insights are possible because the studies are conducted within a single global network in which standardized procedures and immune response assays are utilized. Information gleaned from these insights could accelerate the development of vaccines for HIV, and may be ap-plied to vaccine research for other disease areas as well.

The authors would like to thank Marnie Elizaga, Yunda Huang, Georgia Tomaras, HVTN 044, 052, 055, 060, 063, 065, 068, 070, 071, 076, 077, 078, 080, 087, 088, 094, 097, 098, 204, 205/908 Protocol Teams and participants.

Tracey Day is HVTN Senior Science Writer, Barbara Metch is Senior Statistical Analyst working with the HVTN, Nicole Frahm is Associate Director, Laboratory Sciences at the HVTN Laboratory Program, Cecilia Morgan is HVTN Associate Director, Scientific Development.

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2. Keefer MC, Frey SE, Elizaga M, et al. A phase I trial of preven-tive HIV vaccination with heterologous poxviral-vectors containing matching HIV-1 inserts in healthy HIV-uninfected subjects. Vaccine. 2011;29(10):1948-1958.

3. Goepfert PA, Elizaga ML, Sato A, et al. Phase 1 safety and immunogenicity testing of DNA and recombinant modified vaccinia Ankara vaccines expressing HIV-1 virus-like particles. J Infect Dis. 2011;203(5):610-619.

4. De Rosa SC, Thomas EP, Bui J, et al. HIV-DNA priming alters T cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable. J Immunol. 2011;187(6):3391-3401.

5. Montefiori D, Huang Y, Karuna M, et al. rAd5/NYVAC-B is superior to NYVAC-B/rAd5 and is dependent on rAd5 dose for neutralizing antibody responses against HIV-1. Poster presented at: AIDS Vaccine 2012; September 9-12, 2012; Boston, MA.

6. Bart P, Huang Y, Frahm N, et al. rAd5 prime/NYVAC-B Boost Regimen is Superior to NYVAC-B prime/rAd5 Boost Regimen for Both Response Rates and Magnitude of CD4 and CD8 T-Cell Responses. Poster presented at: AIDS Vaccine 2012; September 9-12, 2012; Boston, MA.

7. Baden LR, Blattner WA, Morgan C, et al. Timing of plasmid cytokine (IL-2/Ig) administration affects HIV-1 vaccine immunoge-nicity in HIV-seronegative subjects. J Infect Dis. 2011;204(10):1541-1549.

8. Kalams SA, Parker S, Jin X, et al. Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults. PLoS ONE. 2012;7(1):e29231.

9. Kaldor J, Edupuganti S, Elizaga M, et al. Robust immunogenic-ity after intramuscular HIV DNA vaccination with IL-12 plasmid cytokine adjuvant delivered via electroporation in HIV uninfected adults (HVTN 070 & 080). Poster presented at: AIDS Vaccine 2011; September 12-15, 2011; Bangkok, Thailand.

10. Morgan C, Peiperl L, McElrath MJ, et al. DNA prime fol-lowed by adenoviral vector boost elicits HIV-1 specific CD8+ T cell responses in healthy HIV-1 uninfected adults (HVTN 052 and 057). In: AIDSVaccine 2007; August 20-23, 2007; Seattle, WA. Abstract P06-15.

11. Churchyard GJ, Morgan C, Adams E, et al. A phase IIA randomized clinical trial of a multiclade HIV-1 DNA prime fol-lowed by a multiclade rAd5 HIV-1 vaccine boost in healthy adults (HVTN204). PLoS ONE. 2011;6(8):e21225.

12. Morgan C, Jin X, Yu X, et al. DNA plasmid HIV vaccine design,


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number of doses, participant gender, and body mass index affect T-cell responses across HIV vaccine clinical trials [Abstract]. Retrovirol-ogy. 2012; 9 (Suppl 2):P133.

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18. Horton H, Thomas EP, Stucky JA, et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods. 2007;323(1):39-54.

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The HVTN’s mission is to drive, as rapidly and scientifi-cally rigorously as possible, the development of safe and effective vaccines for prevention of HIV infection glob-ally. To that end, the Network must develop tools that enable it to answer the critical questions in this rapidly evolving field. These tools include novel clinical trial designs, from early phases through efficacy studies.

In a previous HVTNews article,1 Gilbert and Grove described innovative sequential designs for HIV vac-cine efficacy trials in which the primary objective is the assessment of vaccine efficacy in reducing the rate of HIV infection (see also Gilbert, Grove, et al., 2011).2 But what about the design of pre-efficacy trials, where the primary objectives involve safety and immunogenicity assessments? As some traditional design strategies, used in other clinical trial settings, may not be feasible in the context of HIV vaccine evaluations, innovative statistical designs are particularly important to HVTN trials.

There are 2 major goals for the HVTN’s agenda in early phase, pre-efficacy trials. One is first-in-human safety and immunogenicity evaluation of vaccine candidates. The other is selection of vaccine regimens to move onto the next level of testing. For the former, often only a relatively small number of participants is needed to establish initial vaccine safety. In dose escalation studies, for example, 10 participants are often evaluated at each dose level to determine the maximally tolerated dosage (MTD). For further characterization of the vaccine’s safety and immunogenicity, additional subjects (n=15-30) are enrolled at the MTD. Such a design provides a reasonably high probability of moving a truly promising vaccine candidate forward, or stopping the study of a weak one, with a low chance of missing a promising vac-cine candidate or wrongly selecting a weak one. An im-munogenicity checkpoint can be instituted; for example, if ≥ 3 positive responses are seen out of 25 at the MTD, continue evaluation, else (ie, 0-2 out of 25) stop. The

principle of such stopping rules is to stop only if some maximum immune response rate can be ruled out with high probability, while the choice of maximum immune response rate often depends on the portfolio of candi-date vaccine regimens being evaluated. When feasible, multiple vaccine products at similar stages of develop-ment from different vaccine developers may be tested in the same study, sharing common placebo samples and reducing trial development overhead.

Once initial safety and immunogenicity are established, there may be interest in further regimen optimization among more than 2 regimens in terms of immunoge-nicity. When it is not critical to establish superiority or non-inferiority of one vaccine candidate compared to the others, and the study vaccine regimens and objectives do not lend themselves to a more structured statistical de-sign, such as factorial or Latin square (discussed below), a rank-and-select design can be an efficient approach to advancing regimens.3 Such a design often ranks all active comparator arms and selects the arm or arms with the best immune response, such as response rates and/or response magnitudes. Rank-and-select designs in general require a smaller sample size than superiority and non-inferiority trials. A rank-and-select design may be useful, for example, in a dose de-escalation study of an adjuvant, to evaluate the optimal adjuvant dose for eliciting a cer-tain immune response.

The above mentioned approaches have been imple-mented in multiple HVTN trials. As the HIV vac-cine field continues to mature, there are currently more HIV vaccine products available for testing in various combinations than ever before. For example, there are different DNA constructs, viral vectors, vector inserts of HIV genes, and proteins. Furthermore these products could also be combined with different types of adju-vants, given at different dose levels, and administered through various routes and delivery methods. There are

Can We Be More Effective in Designing Pre-efficacy Vaccine Trials?Yunda Huang, Zoe Moodie, and Cecilia Morgan


CAN We Be moRe eFFeCTiVe iN DesigNiNg PRe-eFFiCACY VACCiNe TRiAls?

also different factors that may play an important role in vaccine evaluation, including demographics, host genet-ics, and environmental factors such as prior exposure to the vaccine vector, co-infection with other pathogens, or background use of non-vaccine prevention modalities. In selecting the best regimen(s) for the next stage of test-ing, one primary challenge comes from this vast number of combinations that must be taken into account in an early-phase trial design.

From a trial design perspective, such challenges may be ameliorated by more systematic, structured evaluations of these different types of factors. One example of a sys-tematic structured design is the factorial design, where more than 1 factor with multiple levels can be studied simultaneously. In a factorial design, study participants

are randomized across combinations of factor levels, each representing 1 vaccine regimen of interest for evaluation. As an example, a 2 × 2 factorial design could be used to randomize participants into 1 of 4 groups defined by 2 novel adjuvants combined with 2 vaccine regimens such as a DNA + viral vec-tor + protein vs a viral vector + protein (see Figure 1). Multiple questions can be addressed in such a factorial trial including: (1) How do the adjuvants differentially impact immuno-genicity? (2) How does the DNA component impact immunogenicity? (3) Does the adjuvant effect differ with and without DNA? (4) Does the DNA effect differ between the adjuvants? In contrast, to obtain the same precision for estimating main effects of each factor, single-factor designs would require twice as many subjects, while only being able to answer the first 2 ques-tions.

The biggest advantage of using a factorial design relies on the assumption that results about any given factor can be combined over the levels of the other factors for the assessment of main effects. However, care is needed in factorial designs when antagonism may occur with combinations of products. Large sample sizes are needed to assess the effect of interactions, and the power to as-sess the main effects may be compromised by negative interactions.

When certain factors are not of particular interest for direct comparisons, but can potentially influence immu-nogenicity, randomized block designs, which randomize treatments within blocks of participants, can be used to systematically control for variability arising from spe-cific factors, and improve efficiency when the blocking

factor(s) predict immunogenicity. Instead of randomizing across all combinations of factors, participants are not randomized across the blocking factors. Therefore, caution is needed in the interpretation of main effects of (non-randomized) blocking factors, and interactions between randomization and blocking factors. The optimally efficient randomized block

design for detecting main effects of treatment is the Latin squares design, which minimizes nui-

sance sources of variability in immunogenicity outcomes by systematic blocking in 2 directions. For example, the replicated Latin squares design shown in Figure 2

evaluates 3 vaccine candidates -- A, B, and C -- under different types of pre-exposure prophylaxis (PrEP) usage (oral, topical, and combination) and different delivery methods of the vaccine (intramuscular by needle (IM), intramuscular by electroporation (EP-IM), and intrader-mal by needle (ID). Instead of being randomized into 27

roW FacTor:

deliverY meTHod

columN FacTor: PreP usaGe

ORAL TOPICAL COMBINATION

IM a (n=30) b (n=30) C (n=30)

Ep-IM C (n=30) a (n=30) b (n=30)

Id b (n=30) C (n=30) a (n=30)

Figure 2. 3x3 Latin square design example

FacTor B:

+/- dNa iN reGimeN

FacTor a: adJuvaNTs

ADjuVANT A ADjuVANT B

dNa + Viral Vector + protein N=25 N=25

Viral Vector + protein N=25 N=25

Figure 1. 2x2 Factorial design example


categories (3x3x3, determined by PrEP usage, delivery method, and vaccine) as a factorial design would, study participants falling into any of the 9 combinations of PrEP usage and delivery method are randomized to re-ceive 1 of the 3 tested vaccines. This design can answer 3 questions: (1) Which vaccine works best overall? (2) For each given vaccine delivery method or PrEP usage type, which vaccine is best? (3) Does vaccine delivery method or PrEP usage type affect immunogenicity?

This design, however, cannot be used to assess inter-actions of vaccine regimen with the row and column factors; thus the niche for this design is in an assessment of vaccine effects when it is not critical to assess interac-tions.

The HVTN has entered into a very exciting, yet chal-lenging, era where many interesting vaccine concepts are available, and more novel options are forthcoming. Innovative designs in early phase trials can improve efficiency in evaluating the large number of vaccine candidates (Table 1). To fully utilize the advantages that these and other innovations afford us, continued close collaboration among a range of experts including researchers, community leaders, and biostatisticians is

needed, so that the Network can carry out its important task of moving the field forward, and working towards finding an effective HIV vaccine.

Yunda Huang and Zoe Moodie are Senior Staff Scientists and statisticians, Statistical Center for HIV/AIDS Research and Prevention (SCHARP), working with the HVTN; Cecilia Morgan is HVTN Associate Director, Scientific Development.

Reference List

1. Gilbert P, Grove D. A sequential two-stage trial design for evaluating efficacy and immune correlates for multiple vaccine regi-mens. HVTNews. 2011;3(1):2-4.

2. Gilbert PB, Grove D, Gabriel E, et al. A sequential phase 2b trial design for evaluating vaccine efficacy and immune correlates for multiple HIV vaccine regimens. Statistical Communications in Infectious Diseases. 2011;3(1), Article 4.

3. Moodie Z, Rossini AJ, Hudgens MG, Gilbert PB, Self SG, Russell ND. Statistical evaluation of HIV vaccines in early clinical trials. Contemp Clin Trials. 2006;27(2):147-160.

CAN We Be moRe eFFeCTiVe iN DesigNiNg PRe-eFFiCACY VACCiNe TRiAls?

PlausiBiliTY oF iNTeracTioNs

iNTeresT iN iNTeracTioNs

iNTeresT iN maiN eFFecTs

# oF FacTors, # oF TreaTmeNTs

recommeNded desiGN examPles

High High High >=2, >=4 Factorial designs powered to examine interactions

dNa x viral vector (with or without protein)

Medium/Low Low High for vaccine >=2, >=4 Factorial designs only pow-ered to examine main effect

Vaccine x adjuvant

Medium/Low Low High for treatment;Low for blocking

factors

=2, >=3 replicated Latin square designs

Vaccine x host genetics x dose

Low Low Low N/a, >=3 rank-and-select Vaccine regimen comparison

Table 1. Paradigm for selecting an appropriate structured design for phase 1b vaccine trials


2200 STRONGCELEBRATING OUR VOLUNTEERSHVTN CONFERENCE RECEPTION, OCTOBER 29, 2012

At the 2012 HVTN Fall Full Group Meeting in Seattle, the first night’s reception included a special celebration for reaching an enrollment milestone of 2200 participants in HVTN 505. At the time the reception was planned, 2200 was the trial’s target enrollment, and given the importance of this trial (currently the only pre-ventive HIV vaccine efficacy trial worldwide), an acknowledgement of this achievement was in or-der. Special guests at the reception were models from the Seattle HIV Vaccine Trials Unit’s suc-cessful recruitment campaign, which featured local well-known members of the community in ads seeking volunteers for the Unit’s vaccine trials. This strategy has also been used success-fully at several other HVTN sites.


(facing page, left) Blue Bear Wharton (center) poses with Davora Lindner and her partner Ro Yoon of Seattle's HIV Vaccine Trials Unit. Blue Bear as Debrianna modeled for the HVTU's promotional campaign.

(facing page, right) Entertainer and community activist Aleksa Manila looks beautiful for the camera.

(right) Photographer Nate Gowdy poses along-side Glo Euro N'Wei, Yuriko Lomein and Marilyn Moan-roe of the Sisters of Perpetual Indulgence, The Abbey of St. Joan. The Sisters donated their time to help Seattle's HIV Vaccine Trials Unit with outreach and even modeled for the promotional campaign.

(below) Seattle HVTU Outreach Specialist Jacob McIntyre likes the HVTU’s recruitment poster featuring Tanya, Miss U.T.O.P.I.A 2011. (U.T.O.P.I.A seeks to create a safe space for Pacific Islanders LGBTQI communities, advocating for social justice, education, and overall wellness.)

(backgound) Chihuly gardens illuminate the night.

Reception photos: Nate Gowdi; Recruitment Post-ers: Nate Gowdy and Carlos Paradinha.


An exciting milestone for HIV vaccine research has been the identification of antibodies capable of neutralizing diverse HIV strains in chronically infected patients. Although somewhat rare, the existence of these broadly neutralizing antibodies (bnAbs) hints that the immune system is capable of developing antibodies with sufficient cross-reactivity to combat HIV’s extreme variability. In the last several years, new technologies have been applied to bnAb discovery, resulting in a surge of new antibod-ies with even greater neutralization breadth and potency. These bnAbs provide valuable information for guiding immunogen design in classic vaccination approaches.1

Several novel applications are also possible for bnAbs that are manufactured as monoclonal antibodies (mAbs). These are based on the ability to administer mAbs to humans and thereby establish immunity passively. Mul-tiple delivery approaches are under investigation, includ-ing direct administration via infusion or injection, or through vector-mediated gene transfer. Clinical studies using bnAb transfer provide the opportunity to test in humans, for the first time, whether bnAb presence can prevent HIV acquisition. Such proof of concept testing would establish an important immunological benchmark for efficacious neutralizing activity levels. If proven, such studies could lead to the development of bnAb transfer as a strategy for prevention of HIV infection or disease progression. In addition, this would enable the inves-tigation of HIV neutralization mechanisms to inform immunogen design. The HIV Vaccine Trials Network (HVTN) has great interest in moving forward with clinical evaluations of bnAbs because of the power these studies could have to advance HIV vaccine development.

challenges for induction of bnabs against Hiv

During natural HIV infection, HIV-specific antibodies develop during the first several weeks of infection.2 Some of these early antibodies possess neutralizing activity as

measured in vitro, but their reactivity is limited to closely related HIV strains. After several years of infection, a proportion (10-30%) of individuals develops antibodies with broader reactivity, as judged by neutralization of a panel of diverse viral isolates in vitro.3-7 The failure of HIV infected hosts to develop bnAbs in a timely man-ner is due to the numerous evasion strategies employed by the virus. For example, neutralizing antibodies impede viral entry into host cells by targeting the HIV envelope protein (Env), which is hypervariable across virus strains and even among isolates within an infected individual.8,9

Env has multiple unique properties that serve to ef-fectively avoid host bnAb development. The Env spike on the HIV surface is a membrane-bound heterotrimer comprising the glycoprotein subunits gp41 and gp120 (see Figure 1).10 Env conformational instability leads to non-native forms on virion surfaces that misdirect im-mune responses toward non-neutralizing responses.11,12 Several bnAb binding epitopes, such as the CD4 binding site, are buried in the native structure impeding antibody binding.13,14 In addition, glycan residues form a shield on the surface further masking antibody binding epit-opes.15,16 Neutralizing antibodies that develop can target variable regions, and may therefore be strain specific.17

Properties of bnabs targeting Hiv

bnAbs have not yet been induced via vaccination in humans largely due to the challenges posed by Env and described above. Nonetheless, numerous bnAbs have been identified in chronically infected patients and are being used to understand how antibody mediated viral neutralization is accomplished.1,18

Early efforts identified 5 bnAbs targeting 3 different Env epitopes: 2G12 recognizing a glycan-associated epit-ope,19,20 b12 recognizing an epitope in the CD4 binding site,21,22 and 2F5, 4E10, and Z13 recognizing regions

Broadly Neutralizing Monoclonal Antibodies: Powerful Tools for HIV Vaccine DevelopmentTracey Day


BRoADlY NeuTRAliziNg moNoCloNAl ANTiBoDies:

near the membrane attachment site of gp41 (membrane proximal external region, MPER) (see Figure 1).23-26 These antibodies neutralized virus strains from multiple clades in vitro, and possessed a number of unusual fea-tures, such as exceptionally high levels of somatic muta-tion.

During the last several years new technologies have been applied to bnAb research and resulted in a large increase in the number of bnAbs identified. For example, resurfaced glycoprotein probes have been constructed and used to screen sera for epitope-specific neutralizing activity.27-29 Other new methods, such as single B cell cloning, deep sequencing, and high-throughput micro-neutralization assays facilitated the detection and clon-ing of bnAb expressing B cells.30-35 Several of these new bnAbs possess improved characteristics, such as excep-tionally broad neutralization activity (80-100% of tested virus isolates), and vastly increased potency.1

Some of the new bnAbs, such as VRC01, bind to epitopes within the CD4 binding site, but have greater

neutralization breadth than the early antibodies that target this epitope.29 Many of the recently identified bnAbs, however, bind to new epitopes (see Figure 1). For example, 2 clonally related bnAbs, PG9 and PG16, bind to a conformational epitope made up of V1 and V2 loop quaternary structure residues.36 Other examples include PGT121, a member of the V3 loop carbohydrate epitope-specific group.37 Together, these bnAb epitopes reveal multiple vulnerable sites on Env that may be tar-geted with increasing precision for vaccine immunogen design.38

The humanized mouse monoclonal antibody, ibalizumab (TaiMed/Aaron Diamond AIDS Research Center), is an example of alternative concept for HIV neutraliza-tion. This antibody potently blocks entry of diverse HIV strains into host cells by binding to CD4, the primary host cell receptor utilized by HIV.39 Bispecific antibody-like molecules, comprising ibalizumab in combination with other bnAbs described above, have been engineered and shown to achieve remarkably high potency and neu-tralization breadth in vitro.40

Figure 1. Broadly neutralizing antibody (bnAb) binding sites identify vulnerable regions of the HIV Envelope spike that may be targeted for immunogen design. bnAb binding sites have been localized to the V1/V2 loop (PG9, PG16), V3 loop (PG9, PG16), CD4 binding site (b12, VRC01, HJ16), and MPER (2F5, 4E10, Z13e1) regions.

Glycan shield V1-V2 loop V3 loop

C4 binding site MPER

gp120gp41

viral envelopespike


BRoADlY NeuTRAliziNg moNoCloNAl ANTiBoDies

Collectively, these bnAb studies reveal multiple locations that are vulnerable to antibody neutralization. Research-ers are applying this knowledge toward immunogen design in a variety of ways. One strategy is to develop methods to present Env in more native-like or exposed conformations through the use of trimeric proteins or virus-like particles displaying membrane-bound Env on their surface. In addition, studies are being conducted to learn how bnAbs develop in chronically infected patients in hopes of shepherding bnAb development via stepwise vaccinations with increasingly specific immunogens.1 That bnAbs develop in humans indicates the possibility that immunogens may be designed to elicit bnAbs via classic vaccination approaches.

immunization with bnabs

Passive immunization involves the transfer of antibod-ies to achieve temporary or conditional protection from infection. This is in contrast to classic active vaccination approaches, in which pathogen-specific antigen delivery elicits a long lasting protective immune response. Anti-body transfer for passive immunization may be accom-plished by direct administration of mAbs. To maintain sufficient antibody levels for protection against HIV, repeated administrations of broadly neutralizing mAbs (bnmAbs) would be needed, for example on a monthly or quarterly basis. mAb delivery is a well-established platform for immunotherapy against several cancers and in one case to prevent infectious disease (respiratory syncytial virus in infants).

Alternatively, vector-mediated gene delivery can be used to administer bnAb. In this case, the genes encoding the antibody are inserted into a viral vector, for example adeno-associated virus (AAV), and this is used to trans-duce host cells in vivo. This approach renders the person “immunized” so long as the transduced gene is expressed and therefore antibody is produced.

Both direct and vector-mediated approaches have been used to transfer bnAbs to non-human primates. Using different simian HIV (SHIV) challenge models, bnAbs have successfully protected animals from SHIV infection to various degrees.38,41 These findings support the notion

that transfer or induction of bnAbs in humans could reduce HIV infection rates; however clinical studies in humans are needed to verify this concept.

clinical trials with bnab to advance vaccine development

A major outstanding question for HIV vaccine research is whether bnAbs present prior to HIV exposure have the capacity to reduce HIV infection rates in human populations. Passive immunization with the new excep-tionally potent and broadly reactive HIV-specific anti-bodies provides a means to test this concept in human clinical trials.

The goal of the initial studies will be to determine the safety of the bnAb products. Additional aims of early studies with direct bnAb administration will be to define the pharmacokinetic profiles and identify appropriate dosing regimens. For vector-mediated delivery, vector safety and persistence will be primary aims, as well as antibody expression levels and durability.

These initial studies will establish a foundation to enable subsequent studies to define bnAb properties required for efficacy -- for example, the necessary bnAb concen-trations in sera and mucosal sites, and essential Fc–medi-ated effector functions. Identifying these properties will provide critical immunogenicity targets for the field, aiding in the prioritization of vaccine products more likely to achieve efficacy, and facilitating identification of immune correlates of protection.

The HVTN is well-suited to conduct these clinical tri-als with clinic sites experienced in the necessary safety evaluations and studies employing frequent sampling. In addition, the HVTN Laboratory Program has extensive expertise in antibody evaluations in sera and mucosal specimens.

The discovery of ever more potent and broadly reactive antibodies allows investigators to evaluate these bnAbs in clinical trials. Such trials can provide the critical proof of concept for the utilization of bnAbs in preventing HIV acquisition in humans. These trials can also sup-



ply the HIV vaccine field with a wealth of information for vaccine immunogen design. Knowledge gained in conducting these assessments will facilitate the fulfill-ment of the HVTN’s mission of developing, as rapidly as possible, a safe and effective vaccine for prevention of HIV infection globally.

The author wishes to thank Cecilia Morgan for helpful dis-cussions.

Tracey Day is Senior Science Writer at the HVTN.

References1. Bonsignori M, Alam SM, Liao HX et al. HIV-1 antibodies from infection and vaccination: insights for guiding vaccine design. Trends Microbiol. 2012;20(11):532-539.

2. Tomaras GD, Yates NL, Liu P, et al. Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia. J Virol. 2008;82(24):12449-12463.

3. Mikell I, Sather DN, Kalams SA, Altfeld M, Alter G, Stamata-tos L. Characteristics of the earliest cross-neutralizing antibody response to HIV-1. PLoS Pathog. 2011;7(1):e1001251.

4. Mascola JR, D'Souza P, Gilbert P, et al. Recommendations for the design and use of standard virus panels to assess neutralizing an-tibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J Virol. 2005;79(16):10103-10107.

5. Deeks SG, Schweighardt B, Wrin T, et al. Neutralizing antibody responses against autologous and heterologous viruses in acute versus chronic human immunodeficiency virus (HIV) infection: evidence for a constraint on the ability of HIV to completely evade neutralizing antibody responses. J Virol. 2006;80(12):6155-6164.

6. Gray ES, Moore PL, Choge IA, et al. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection. J Virol. 2007;81(12):6187-6196.

7. Doores KJ, Burton DR. Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16. J Virol. 2010;84(20):10510-10521.

8. Stamatatos L, Morris L, Burton DR, Mascola JR. Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine? Nat Med. 2009;15(8):866-870.

9. Korber B, Gaschen B, Yusim K, Thakallapally R, Kesmir C, De-tours V. Evolutionary and immunological implications of contempo-rary HIV-1 variation. Br Med Bull. 2001;58:19-42.

10. Schief WR, Ban YE, Stamatatos L. Challenges for structure-based HIV vaccine design. Curr Opin HIV AIDS. 2009;4(5):431-440.

11. Zhu P, Chertova E, Bess J, Jr., et al. Electron tomography analy-sis of envelope glycoprotein trimers on HIV and simian immunode-ficiency virus virions. Proc Natl Acad Sci U S A. 2003;100(26):15812-15817.

12. Poignard P, Moulard M, Golez E, et al. Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies. J Virol. 2003;77(1):353-365.

13. Myszka DG, Sweet RW, Hensley P, et al. Energetics of the HIV gp120-CD4 binding reaction. Proc Natl Acad Sci U S A. 2000;97(16):9026-9031.

14. Chen L, Kwon YD, Zhou T et al. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science. 2009;326(5956):1123-1127.

15. Wyatt R, Kwong PD, Desjardins E et al. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature. 1998;393(6686):705-711.

16. Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape by HIV-1. Nature. 2003;422(6929):307-312.

17. Phogat S, Wyatt R. Rational modifications of HIV-1 en-velope glycoproteins for immunogen design. Curr Pharm Des. 2007;13(2):213-227.

18. Kwong PD, Mascola JR. Human antibodies that neutralize HIV-1: identification, structures, and B cell ontogenies. Immunity. 2012;37(3):412-425.

19. Calarese DA, Lee HK, Huang CY, et al. Dissection of the car-bohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc Natl Acad Sci U S A. 2005;102(38):13372-13377.

20. Buchacher A, Predl R, Strutzenberger K, et al. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lympho-cyte immortalization. AIDS Res Hum Retroviruses. 1994;10(4):359-369.

21. McInerney TL, McLain L, Armstrong SJ, Dimmock NJ. A hu-man IgG1 (b12) specific for the CD4 binding site of HIV-1 neutral-izes by inhibiting the virus fusion entry process, but b12 Fab neutral-izes by inhibiting a postfusion event. Virology. 1997;233(2):313-326.

22. Roben P, Moore JP, Thali M, Sodroski J, Barbas CF, III, Burton DR. Recognition properties of a panel of human recombinant Fab



fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J Virol. 1994;68(8):4821-4828.

23. Muster T, Steindl F, Purtscher M, et al. A conserved neutraliz-ing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 1993;67(11):6642-6647.

24. Conley AJ, Kessler JA, Boots LJ, et al. Neutralization of diver-gent human immunodeficiency virus type 1 variants and primary isolates by IAM-41-2F5, an anti-gp41 human monoclonal antibody. Proc Natl Acad Sci U S A. 1994;91(8):3348-3352.

25. Stiegler G, Kunert R, Purtscher M et al. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses. 2001;17(18):1757-1765.

26. Zwick MB, Labrijn AF, Wang M, et al. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol. 2001;75(22):10892-10905.

27. Corti D, Langedijk JP, Hinz A, et al. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS ONE. 2010;5(1):e8805.

28. Scheid JF, Mouquet H, Ueberheide B, et al. Sequence and struc-tural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011;333(6049):1633-1637.

29. Wu X, Yang ZY, Li Y, et al. Rational design of envelope identi-fies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010;329(5993):856-861.

30. Doria-Rose NA, Klein RM, Manion MM, et al. Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies. J Virol. 2009;83(1):188-199.

31. Scheid JF, Mouquet H, Feldhahn N, et al. A method for iden-tification of HIV gp140 binding memory B cells in human blood. J Immunol Methods. 2009;343(2):65-67.

32. Scheid JF, Mouquet H, Feldhahn N, et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infect-ed individuals. Nature. 2009;458(7238):636-640.

33. Traggiai E, Becker S, Subbarao K, et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004;10(8):871-875.

34. Kwong PD, Mascola JR, Nabel GJ. Mining the B cell repertoire for broadly neutralizing monoclonal antibodies to HIV-1. Cell Host Microbe. 2009;6(4):292-294.

35. Simek MD, Rida W, Priddy FH, et al. Human immunodeficien-cy virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutral-ization assay together with an analytical selection algorithm. J Virol. 2009;83(14):7337-7348.

36. Walker LM, Phogat SK, Chan-Hui PY, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326(5950):285-289.

37. Walker LM, Huber M, Doores KJ, et al. Broad neutraliza-tion coverage of HIV by multiple highly potent antibodies. Nature. 2011;477(7365):466-470.

38. Mascola JR, Montefiori DC. The role of antibodies in HIV vac-cines. Annu Rev Immunol. 2010;28:413-444.

39. Pace CS, Fordyce MW, Franco D, Kao CY, Seaman MS, Ho DD. Anti-CD4 Monoclonal Antibody Ibalizumab Exhibits Breadth and Potency Against HIV-1, With Natural Resistance Mediated by the Loss of a V5 Glycan in Envelope. J Acquir Immune Defic Syndr. 2013;62(1):1-9.

40. Pace CS, Song R, Franco D, Seaman MS, Ho DD. PG9-ibalizumab and VRC01-ibalizumab bispecific fusion antibodies exhibit essentially 100% breadth, picomolar potency and synergistic mechanism of action. Poster presented at: AIDS Vaccine Conference – Bangkok; September 12-15, 2011; Bangkok, Thailand.

41. Pantophlet R, Burton DR. GP120: target for neutralizing HIV-1 antibodies. Annu Rev Immunol. 2006;24:739-769.


Seattle HVTu Helps Pilot HANC Native American Initiativejessica Velcoff, Katie Osterhage, Michael Arnold, Robert Foley, Damon Humes, jeffrey Schouten, and Adi Ferrara

In line with its commitment to engage communities highly impacted by HIV/AIDS, the HVTN, through its Seattle clinical research site (CRS), took part in a novel pilot project starting in the fall of 2011. The project, Native American Engagement in HIV Clinical Research (NAEHCR, pronounced “nature”), is aimed at engaging urban Native communities in HIV clinical research. NAEHCR was developed with recognition that the health and well-being of Native individuals and communitiesa could greatly benefit from raised aware-ness and involvement in such research. The Seattle site was chosen, in part, because the Pacific Northwest has a concentrated urban Native population.

Native communities in the US face challenges to en-gagement in clinical research similar to other popula-tions that are often under-represented in clinical trials. A reluctance to participate stems from historical trauma1 due to a legacy of poorly conducted medical research. Moreover, many Native people are frustrated with ‘heli-copter’ research, and advocate for respectful research that carries with it long-term benefit to their communities. According to one Native participant, researchers tend to “come in, they do their research, and then they leave and they don’t tell us what happened. Or it doesn’t benefit our community at all. They just take the things and that still happens.”

Native communities are also highly impacted by the HIV/AIDS epidemic. Native Hawaiian and other Pacific

Islanders, and American Indians and Alaska Natives (AI/AN) have the 3rd and 4th highest rates of new HIV diagnoses among all racial/ethnic groups, respectively. Moreover, AI/ANs have the highest percent increase of new HIV infections, increasing by 8.7% from 2007 to 2010, and the highest mortality rate after an AIDS diagnosis.2

NAEHCR is an innovative partnership between the Office of HIV/AIDS Network Coordination’s Legacy Project and the National Native American AIDS Pre-vention Center, with funding from the NIH’s Division of AIDS. The Denver CRS of the AIDS Clinical Trials Group (ACTG) served as the second site for the pilot. Also participating was the Seattle AIDS Clinical Trials Unit (ACTU). Site staff worked collaboratively with Na-tive Community Consultants (NCCs, groups of Native leaders and advocates) to advise the project, with a goal of enhancing the participating sites’ community engage-ment (CE) activities with urban Native communities.

The importance of the project was evident to everyone involved. One CRS staff member noted, “I think there’s a lot of mistrust by Native communities and other ethnic communities about clinical research, especially when it’s being done by the US government, because we surely have a long track record of problems that people can point to. So it’s difficult.” An NCC member stated, “The NAEHCR project is important because it provides knowledge, understanding, and help to the Native com-


seATTle HVTu HelPs PiloT HANC NATiVe AmeRiCAN iNiTiATiVe

munity. It also gives us a voice and delivers vital infor-mation back to the medical community.”

During the development phase, NAECHR activities included a) formative research involving Native com-munity members and CRS staff, b) NCC-directed CE activities with identification of challenges and oppor-tunities for promoting and sustaining such activities, c) delivery of cultural humility trainings3 to CRS staff, and d) HIV research literacy trainings to NCCs. In Seattle, the HVTN CRS’s Community Education Coordinator, Ro Yoon, was in charge of leading the site’s engagement with Native communities. She explained that as part of the pilot, the site committed to ensure all staff members received the training. Asked to explain the difference between cultural sensitivity and cultural humility training, Yoon responded that cultural humility training “is an ongoing process that asks the questions about one’s role while participating in the target com-munity.” In other words, the training, instead of teach-ing a participant about the target community, encour-ages them to learn from the target community about the experience of being part of that community.

During the formative research stage, information was collected and analyzed that led to the development of the staff training and CE activities. This research explored awareness and attitudes about local Native communities among CRS staff, and local HIV clini-cal research among Native community members. Key takeaways from this research are presented in Table 1. NAEHCR involved both CRS staff and NCC members throughout the research process to ascertain best prac-

tices for local community engagement. CRS representa-tives and NCCs gave feedback on design, implementa-tion, and interpretation of research findings. Qualitative data was collected via interviews with select CRS staff (n=4) and focus groups with NCCs (n=24) in Denver and Seattle. Surveys were administered at local Native community health and social events (n=115).

The research indicated several key findings that guided CE planning. First, it was evident that CRS staff and Native stakeholders had little interaction and that both groups could benefit from a targeted focus on CE with urban Native communities. As one CRS staff member noted, knowledge about local Native culture was lim-ited: “I don’t know a lot about Native culture in [local] tribes. I do know that this area is rich with Native cul-ture and Native peoples. But again, because we haven’t had much interaction with people coming in here of Native origin my knowledge, I’m sad to say, is minimal.” Relatedly, community surveys indicated that Native communities had limited awareness of their local CRS; only 15 (13%) of the community survey participants had heard of vaccine trials in their city. At the same time, CRS staff indicated a desire to engage local Native communities: “I think learning anything about another culture is important… also getting a better understand-ing of how people view research, since that’s really our work here and what we’re trying to address.” Native community members also indicated support for HIV clinical research, particularly in light of the community benefits of participating in HIV clinical trials: 89 Native community survey participants (78%) indicated that participation in HIV clinical studies would benefit their entire community.


NAEHCR SuMMARY OF MAIN THEMES FROM FORMATIVE RESEARCH

1. Native community engagement Facilitators � Strong sense of community

� Emphasis on community health and wellness

� Successes of other health initiatives

� High level of engagement in HIV related activities

� Moderately positive attitudes towards clinical research

(particularly vaccine trials)

2. Native community-level Barriers to Participation in research

� Historical trauma

� Mistrust in research

� Lack of reciprocity

3. Native community-level Barriers to Hiv clinical research

� Stigmatization of people living with HIV/AIDS

� Stigmatization of LGBT/ Two-Spirit1 community

� Silence around HIV

� Dearth of HIV resources for Native communities

� Low awareness of HIV clinical research

4. Hiv clinical research site dynamics � Low involvement of Native people

� Variable knowledge of and experience with Native communities

� General difficulties in recruiting in non-white communities

� Lack of institutional resources

� Interest in NAEHCR Project

5. Factors to consider in developing engagement strategies

� Consistency and visibility of research staff

� Native representation among service providers

� Intergenerational involvement among community

� Engagement across genders within community

1 Two-Spirit is an identity that embodies the belief among some tribes that people can carry both feminine and masculine spirits. It can also be used to describe Native LGBT individuals, who in many Native tribes were honored – prior to colonization. Today, there is a social/political movement to reclaim varying positions that many individuals of 'non-traditional' gender and social roles historically held among many tribes.

Findings also indicated that there are opportunities for CE with urban Native communities. As one focus group participant noted, Native communities tend to place high value on community health and wellness, “So we nip it (health issues) in the bud before it goes higher. Then we have helped ourselves, [our] community, and future generations.” Survey participants indicated moderate involvement in HIV-related activities, with 54% indicat-ing that they had been tested for HIV in the past year (n=62) and 61% indicating that they had talked to health professional about HIV in the past year (n=70). Survey participants also expressed interest in increasing aware-ness and connection to CRS, with 89% agreeing that there was need for more information on HIV vaccine research (n=100). As one focus group participant noted: “I think the objective [of HIV clinical research] is good and that’s why I’m here. I’m here to help.”

These results also informed the activities and lessons contained within the cultural humility trainings that were piloted with the CRS staff in Denver and Seattle. The four-hour trainings were designed to increase staff ’s competency for respectful engagement of local Native communities, and serve as a catalyst for the development of long-term, appropriate, and bi-directional relation-ships between the CRSs and these communities.

Ro Yoon explained that one important takeaway from the humility training conducted for the Seattle site was a change in focus that could apply to recruitment efforts in general. Instead of asking “How can we bring people in [to volunteer]?” the better question to ask would be “Why are they not coming in?” The question allows recruiters to relate to the target community’s sensitivities

Table 1.



and specific barriers, and address them from a position of understanding and empathy. As Noel Williams, a clinic assistant at the Seattle CRS, commented after the train-ing, “This is a population that has been taken advantage of in the name of research. It is not surprising that these folks would rather not participate.”

In 2012, the NAEHCR Project received continued funding from DAIDS to support ongoing facilitation of linkages between CRSs and local Native communities through the development of site-specific engagement plans, and to refine the cultural humility training. With this continued funding, the NAEHCR Project expanded the project to Chicago, Illinois (2 sites: 1 ACTU and 1 HVTN) and San Francisco, California (2 sites: 1 ACTU site and 1 multiple network site – HVTN, HIV Preven-tion Trials Network, and the Microbicide Trials Net-work).

NAECHR is continuing to facilitate CE activities in Seattle and Denver, and has begun the formative work in Chicago and San Francisco. If you reside in any of the demonstration cities, keep an eye out, as NAEHCR will be celebrating National Native HIV/AIDS Awareness Day on March 20, 2013.

For more information about the project, please contact Katie Osterhage (Project Lead, [email protected]).

The authors would like to thank Joann Spencer (Navajo, Southern Cheyenne) and Charlene Irani (Kiowa, Seminole) for their contributions to this article, along with all other NCC members that have shaped this project. Addition-ally, we would like to thank our Seattle and Denver CRS

liaisons, Ro Yoon, Michael Louella, and Liam Burghardt along with all other participating site staff. Finally, we would like to thank our funders for their support. The HANC project is funded in whole or in part with Federal funds from the Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Depart-ment of Health and Human Services, grant number 5 U01 AI068614, entitled Leadership Group for a Global HIV Vac-cine Clinical Trials (Office of HIV/AIDS Network Coordi-nation) with additional support from the National Institute of Mental Health.

Jessica Velcoff is a Consultant for the NAEHCR Project; Ka-tie Osterhage is Project Lead, NAEHCR ; Michael Arnold is Social Scientist, NAEHCR Project; Robert Foley is Presi-dent/CEO of NNAAPC; Damon Humes Director, Legacy Project; Adi Ferrara is Technical Editor, HVTN Core.

aNative communities include American Indians, Alaska Natives, Native Hawaiians, and First Nations.

Reference List1. Brave Heart MY, DeBruyn LM. The American Indian Holo-caust: healing historical unresolved grief. Am Indian Alsk Native Ment Health Res. 1998;8(2):56-78.

2. Centers for Disease Control and Prevention, CDC. HIV Sur-veillance Report, 2010, Vol. 22. Department of Health and Human Services, Centers for Disease Control and Prevention; 2012.

3. Tervalon M, Murray-Garcia J. Cultural humility versus cultural competence: a critical distinction in defining physician training outcomes in multicultural education. J Health Care Poor Underserved. 1998;9(2):117-125.



Perspectives from Native Community ConsultantsMy name is Joann Spencer; I’m Navajo and Southern Cheyenne, proud mother of 4 kids. In 2004, I tested positive for HIV. I knew nothing about HIV/AIDS and was always too ashamed to ask questions. I be-came depressed and single with no self-esteem, I heard about a women support group BABES NETWORK Women living with HIV/AIDS. Soon I was there every week. Our support leader Pat handed me a flyer one day and it talked about spreading awareness about HIV/AIDS in the Native Communities -- they needed a person living with HIV/AIDS to share their experience. I really didn’t know what to expect when I got on board with the Legacy Project. At our first meeting, I was pretty intimidated with everyone, because they were using long words I didn’t know. I opened my mind and heart, embracing and educating myself about HIV/AIDS. I have learned so much, it’s made me a stronger woman and I get to share my experience with others, in hopes they learn something or pass the word along. There are some who are still afraid or choose not to lis-ten to our stories. My children are so proud of me and they themselves are no longer afraid nor ashamed of me or for me. My daughters are a part of the project too. I’m very thankful to Jessica, Damon, Katie, Ro, Michael, and the rest of our group on the project; they have made an impact on my life for the better. I’m very thankful for my life.

Joann Spencer is an NCC member from Seattle

I became a member because it is time for me to give back to the community. I am an elder. I went to a wom-an's healing getaway by NNAAPC [National Native American AIDS Prevention Center] and knew I had to get involved. I learned so much in those few days in a non-threatening way. I learned something of myself from what they called historical trauma. I realized that by being a member, I could be part of something so much needed in our community and for myself. I love it. I learn something new every time we meet. I meet others wanting to be part of this fabulous organization as well. It has helped me help others, who have come to me, asking for help after being diagnosed with HIV and I could answer but more importantly provide them a place to get the answers and help they needed. It felt good to be trusted. I felt great that I was able to help another. I love being part of an organization promoting a change of attitude, prevention, understanding and ways to inform our community to become more aware and healthier.

I hope the project will change the way we treat and look at those with HIV. Remove the stigma and pro-vide us with ways to create a healthier, compassionate, and more informed community. Knowledge is power. Togetherness is strength. With the guidance of NNAAPC/NAEHCR, we can create a better, healthier and brighter future for generations to come and I want to be part of that change. I want to see it so I'll be there.

Charlene Irani is an NCC member from Denver

EDITOR-IN-CHIEF: CECILIA MORGAN

MANAGING EDITOR: ADI FERRARA

SENIOR SCIENCE WRITER: TRACEY DAY

PRODuCTION MANAGER: COuRTNEY LIEBI

ILLuSTRATIONS & LAYOuT: LISA DONOHuE

FOR MORE INFORMATION, VISIT: hvtn.org

THaNk You To:

Gail Broder, Kim Louis, jim Maynard, Genevieve Meyer

commeNTs/QuesTioNs?

[email protected]

Tel: 206 667-6300

Fax: 206 667-6366

HVTN/FHCRC, 1100 Fairview Ave North, E3-300

Seattle, Washington 98109-1024

For curreNT aNd arcHived ediTioNs oF HvTNeWs, Please visiT us Here -

hvtn.org/science/hvtnews.html

MISSION OF THE HVTNTo eNHANCe THe DisCoVeRY AND DRiVe THe DeVeloPmeNT oF A sAFe AND gloBAllY eFFeCTiVe VACCiNe To PReVeNT HiV. We do this through well-designed clinical research trials that objectively and ethically address the critical questions of the field.

our objective clinical trial platform lets us evaluate safety, immunogenicity and efficacy of candidate vaccines, as well as design clinical trials that will provide clues on ways to enhance the effectiveness of new vaccines.

We promote the use of new innovations, which may help us get closer to a safe and effective vaccine more quickly.

The HIV Vaccine Trials Network is an international collaboration

of scientists and educators. Support for the HVTN comes from the

National Institute of Allergy and Infectious Diseases (NIAID) of the

u.S. National Institutes of Health, an agency of the u.S. Department

of Health and Human Services. The Network and NIAID have a close,

cooperative working relationship, with shared attention to intellectual

and scientific issues.

©2013 HiV Vaccine Trials Network. All rights reserved.

CALENDAR

coNFereNce oN reTroviruses aNd oPPorTuNisTic iNFecTioNs (croi)MARCH 3-6, 2013Georgia World Congress Center Atlanta, Georgia, uSA

HvTN coNFereNceMAY 7-9, 2013Omni Shoreham Hotel Washington D.C., uSA

sa aids coNFereNcejuNE 18-21, 2013International Convention Centre Durban Durban, South Africa

ias coNFereNce oN Hiv PaTHoGeNesis, TreaTmeNT aNd PreveNTioNjuNE 30-juLY 3, 2013Kuala Lumpur Convention Centre Kuala Lumpur, Malaysia

sPecial aNNouNcemeNTs

As of February 10th, the HVTN sDmC started rolling out changes to the look and organization of the HVTN Atlas web portal. The changes include a new home page with shortcuts to commonly used features, and horizontal tabs to improve site navigation. For questions contact the Atlas team ([email protected]).

We are upgrading the HVTN members’ site to sharePoint in late spring 2013. Training opportunities will be available at and following the HVTN Conference in may. For questions, please contact matthew Kirts, sharePoint Program manager ([email protected]) or Alicia Yamamoto, Project manager ([email protected]).

members of the HVTN Core, lab, and sDmC whose seattle offices were in 1616 eastlake Ave. moved in early september to 1100 eastlake Ave. The new location brings the entire Vaccine and infectious Diseases Division of the Fred Hutchinson Cancer Research Center into the main campus of “The Hutch.”

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