中国在消灭脊髓灰质炎(脊灰)后期使用赛宾株-脊灰灭活疫苗替代口服脊灰减毒活疫苗策略的前瞻性分析

目的 探讨中国在消灭脊髓灰质炎(脊灰)后期的免疫策略.方法 运用循证医学的方法,检索国内外研究资料,调查中国脊灰疫苗生产和使用现状,从免疫策略比较赛宾株-脊灰灭活疫苗(Sabin-Inactivated Poliovirus Vac-cine,slPV)替代口服脊灰减毒活疫苗(Oral Poliomyelitis Live Vaccine,OPV)的可行性,并从免疫程序等方面进行前瞻性综合分析.结果 中国应考虑选择以IPV替代OPV的免疫策略,且国产sIPV即将问世,在政策、技术、疫苗供应和其它保障方面均具备了替代的条件,常规免疫可参照目前OPV免疫程序.结论 中国应进一步加强消灭脊灰后期免疫策略的研究,积极做好sIPV替代OPV的准备工作.     

脊髓灰质炎疫苗应用现状及免疫策略进展

1988年,全球消灭脊髓灰质炎（脊灰）倡议行动启动以来,取得了重大进展。2012年,全球报告脊灰223例,较2011年减少〉60%,本土脊灰流行国家减少为尼日利亚、巴基斯坦和阿富汗,脊灰野病毒（Wild Poliovirus,WPV）病例数下降到历史最低水平。但WPV传播仍未被阻断,无脊灰国家/地区仍面临输入WPV的风险。同时,有些国家正面临使用口服脊灰减毒活疫苗（Oral Poliomyelitis Attenuated Live Vaccine,OPV）所致疫苗衍生脊灰病毒的风险。目前,不同国家/地区评估各自的脊灰发病风险,依据OPV、脊灰病毒灭活疫苗（Inactivated Poliovirus Vaccine,IPV）的风险和收益,不同国家/地区采用不同的免疫策略：仅使用IPV、序贯使用IPV/OPV和仅使用OPV。2013年,世界卫生组织《全球消灭脊灰终结战略计划》中提出,2014年全球阻断WPV传播,2015年所有国家应至少使用1剂IPV,停用OPV中的Ⅱ型组分;2018年完成消灭WPV证实后,停用OPV。现对OPV和IPV的应用现状以及免疫策略进行简述。     

中国脊髓灰质炎疫苗使用历史回顾及免疫策略调整建议

在消灭脊髓灰质炎（脊灰）过程中,脊灰疫苗发挥了重要作用。口服脊灰减毒活疫苗（Oral Poliomyelitis Attenuated Live Vaccine,OPV）是一种安全有效的疫苗,中国于20世纪60年代推广使用OPV,常规免疫接种率逐步提高到〉90％。从1990年开始,部分省（自治区、直辖市）开展了OPV补充免疫活动（Supplementary Immunization Activity,SIA）;1993～2000年,开展消灭脊灰的国家免疫日活动,中国所在的世界卫生组织（World Health Organization,WHO）西太平洋区于2000年实现无脊灰的目标,继续在适龄儿童中加强OPV常规免疫和开展SIA。根据WHO制定的（（2013—2018年消灭脊灰终结战略计划》,2015年所有国家要引进至少1剂灭活脊灰病毒疫苗（Inactivated Poliovirus Vaccine,IPV）,2016年中期使用二价OPV（I＋Ⅲ型）,2018年停用OPV。为确保OPV成功转换IPV,中国应建立部门间协作机制,加快国产IPV研发生产进程,组织开展OPV和IPV转换的相关研究。     

脊髓灰质炎疫苗序贯免疫程序的研究与应用进展

脊髓灰质炎(脊灰)是由脊灰病毒引起的急性肠道传染病,传播广泛,对儿童的健康和生命危害极大。口服脊灰减毒活疫苗(Oral Attenuated Poliovirus Vaccine,OPV)是全球消灭脊灰行动的首选疫苗,已使II型脊灰野病毒于1999年在全球消灭;但在极少情况下会发生疫苗相关麻痹型脊灰(Vaccine-associated Paralytic Poliomyelitis,VAPP)和疫苗衍生脊灰病毒(Vaccine-derived Poliovirus,VDPVs)病例。为了避免这些风险,许多国家常规免疫规划已不再单独使用OPV,而是改用脊灰灭活疫苗(Inactivated Polio Vaccine,IPV)。这些国家在OPV转换成IPV期间,大部分采用了先接种1~2剂IPV,再接种≥2剂OPV的序贯免疫程序。IPV-OPV序贯免疫方案可减少或预防VAPP,同时又能保持较高的体液免疫和肠道黏膜免疫力,能更好地阻断脊灰野病毒在自然界的循环。根据WHO建议,常规免疫规划中只使用OPV的国家应当调整脊灰免疫策略,引进至少1剂次IPV,采取IPV-OPV序贯免疫策略或联合使用OPV和IPV的策略。相信这些转变将会加快全球消灭脊灰野毒株的进程,对实现全球最终根除脊灰的目标具有重要的意义。     

灭活脊髓灰质炎病毒疫苗和口服脊髓灰质炎减毒活疫苗不同免疫策略经济学评价的系统综述

目的系统综述灭活脊髓灰质炎(脊灰)病毒疫苗(Inactivated Poliovirus Vaccine,IPV)和口服脊灰减毒活疫苗(Oral Poliomyelitis Attenuated Live Vaccine,OPV)不同免疫策略经济学评价方法的研究现状。方法检索数据库中所有使用经济学评价方法,比较两种脊灰疫苗免疫策略的文献并进行系统综述。结果共纳入12篇文献。关于脊灰疫苗不同免疫策略的经济学评价研究尚不规范,尤其成本构成的界定差别较大,缺乏疾病流行数学模型的应用,以及效果指标判定阈值的研究。不同经济水平国家或地区使用IPV替代OPV免疫策略是否具有成本效果优势,纳入文献结论存在不一致性。结论脊灰疫苗免疫策略的经济学评价是动态、复杂的过程,评价我国免疫规划引入IPV不同免疫程序的成本效果很有意义。     

脊髓灰质炎流行现状及控制对策

脊髓灰质炎（脊灰）是由脊灰病毒引起的急性肠道传染病,人是脊灰病毒的唯一宿主。1988年世界卫生组织提出2000年在全球范围内消灭脊灰,目前仅有阿富汗、印度、尼日利亚、巴基斯坦4个国家有脊灰野毒株本土病例报告。随着无脊灰目标的逐渐实现,口服脊髓灰质炎减毒活疫苗（OPV）的缺点逐渐凸现,如OPV在热带地区效力低,可引起疫苗相关麻痹脊灰（VAPP）及免疫抑制者长期排毒等,因此,继续使用OPV不能最终消灭脊灰。1996年,美国免疫实践咨询委员会推荐通过先使用脊灰灭活疫苗（IPV）再使用OPV的连续接种程序来增加IPV的使用,IPV的大量使用消除了活疫苗病毒的散播,也消除了VAPP。2008年,我国提出脊灰疫苗的免疫策略可借鉴国外成功的经验,逐步采用IPV替代OPV。目前已有学者进行了前瞻性研究,证实在中国以IPV替代OPV是可行的。     

灭活脊髓灰质炎病毒疫苗在中国应用的咨询意见调查

目的从扩大免疫规划（Expanded Program on Immunization,EPI）专家认知,来探讨灭活脊髓灰质炎（脊灰）病毒疫苗（Inactivated Poliovirus Vaccine,IPV）在中国应用的相关问题,为制定脊灰疫苗免疫策略提供参考。方法以人口数多和疫苗需求量大为原则,在全国范围内选取7个省（自治区）,对30名EPI专家进行开放式问卷调查。结果50％的调查对象希望在2015年国家能将IPV纳入EPI,与世界卫生组织提出的{2013～2018年消灭脊灰终结战略计划》时间进度表同步,专家们一致认同在保证疫苗质量的前提下,应尽可能地降低疫苗成本,IPV可接受价格中位数为20元／剂（范围5～50元／剂）。实现IPV国产化势在必行,卫生行政等政府部门应尽快明确中国脊灰疫苗免疫策略和使用时间进度表,疾病预防控制中心依据卫生行政部门制定的免疫策略提供技术指导和支持,疫苗生产企业应加快IPV的研发、生产和上市。结论EPI专家一致赞同随着全球消灭脊灰的进程,中国逐步引入IPV是大势所趋。     

疫苗相关麻痹型脊髓灰质炎对IPV疫苗选择的影响

摘要:在全球消除脊髓灰质炎（脊灰）的进程稳步推进的大背景下,随着口服脊髓灰质炎减毒活疫苗（Oral Polio Vaccine,OPV）的继续使用,疫苗相关麻痹型脊髓灰质炎（Vaccine-associated Paralytic Poliomyelitis,VAPP）越来越受到人们的关注,VAPP在国内外的发病情况不容忽视。WHO估计全球每年约有250～500例VAPP发生,国内的估算数字是76～161例,其中首剂次疫苗VAPP发生率较高。在疫苗选择上,灭活的脊髓灰质炎疫苗（IPV）最大的优点是能够避免VAPP的发生,但成本效益分析结果并不支持引入IPV。尽管如此,为了消除VAPP的危害,一些发达国家已率先使用IPV疫苗,同时一些发展中国家采用IPV/OPV序贯程序。因此,基于当前我国维持无脊灰的现状,VAPP所带来的公共卫生问题是决定选择IPV疫苗的重要因素,在可承担的价格空间上,IPV疫苗可作为首选疫苗推荐,也可先行引入IPV/OPV序贯程序。     

使用脊髓灰质炎灭活疫苗的必要性及存在的问题

口服脊髓灰质炎（脊灰）减毒活疫苗（Oral Poliomyelitis Attenuated Live Vaccine,OPV）作为全球消灭脊灰的主要手段,在以发展中国家为中心的脊灰流行地区广泛使用。OPV有很多优点,其有效性、安全性、经济性都很显著,但同时也存在由于OPV本身的病毒学特性所引起的各种问题。因此,使用脊灰灭活疫苗（Inactivated Poliovirus Vaccine,IPV）替代OPV的必要性逐渐增加。现就替代OPV的疫苗,尤其是来自减毒的脊灰病毒的IPV,在日本及发展中国家使用的可能性和存在的问题加以论述。     

脊髓灰质炎疫苗接种现状与进展

脊髓灰质炎(脊灰)疫苗可以有效预防脊灰的发生。随着全球消灭脊灰目标的即将实现,后脊灰时代的免疫策略备受关注。本文对脊灰疫苗接种现状、全球及中国消灭脊灰进程及免疫策略等方面进行了综述,探索脊灰疫苗接种由口服脊灰减毒活疫苗(OPV)向灭活脊灰病毒疫苗(IPV)转换过程中可能存在的问题,为长期的免疫策略提供参考依据。     

Intranasal and sublingual delivery of inactivated polio vaccine

Polio is on the brink of eradication. Improved inactivated polio vaccines (IPV) are needed towards complete eradication and for the use in the period thereafter. Vaccination via mucosal surfaces has important potential advantages over intramuscular injection using conventional needle and syringe, the currently used delivery method for IPV. One of them is the ability to induce both serum and mucosal immune responses: the latter may provide protection at the port of virus entry.The current study evaluated the possibilities of polio vaccination via mucosal surfaces using IPV based on attenuated Sabin strains. Mice received three immunizations with trivalent sIPV via intramuscular injection, or via the intranasal or sublingual route. The need of an adjuvant for the mucosal routes was investigated as well, by testing sIPV in combination with the mucosal adjuvant cholera toxin.Both intranasal and sublingual sIPV immunization induced systemic polio-specific serum IgG in mice that were functional as measured by poliovirus neutralization. Intranasal administration of sIPV plus adjuvant induced significant higher systemic poliovirus type 3 neutralizing antibody titers than sIPV delivered via the intramuscular route. Moreover, mucosal sIPV delivery elicited polio-specific IgA titers at different mucosal sites (IgA in saliva, fecal extracts and intestinal tissue) and IgA-producing B-cells in the spleen, where conventional intramuscular vaccination was unable to do so. However, it is likely that a mucosal adjuvant is required for sublingual vaccination. Further research on polio vaccination via sublingual mucosal route should include the search for safe and effective adjuvants, and the development of novel oral dosage forms that improve antigen uptake by oral mucosa, thereby increasing vaccine immunogenicity. This study indicates that both the intranasal and sublingual routes might be valuable approaches for use in routine vaccination or outbreak control in the period after complete OPV cessation and post-polio eradication.     

sIPV process development for costs reduction

Polio is expected to be eradicated within only a few years from now. Upon polio eradication, the use of oral polio vaccines, which can cause circulating and virulent vaccine derived polio viruses, will be stopped. From this moment onwards, inactivated polio vaccines (IPV) will be used for worldwide vaccination against polio. An increased demand for IPV is thus anticipated. As a result, process development studies regarding the IPV production process, developed in the 1960s, have intensified. Studies on yield optimization aiming at costs reduction as well as the use of alternative polio viruses, which are more biosafe for manufacturing, are actual. Here our strategy to setup a new IPV production process using attenuated Sabin polio virus strains is presented. Moreover, aspects on reduction of the costs of goods and the impact of process optimization on sIPV costs are reviewed.     

An inactivated poliovirus vaccine using Sabin strains produced on the serum-free PER.C6® cell culture platform is immunogenic and safe in a non-human primate model

BackgroundThe World Health Organization recommends the development of affordable next-generation inactivated poliovirus vaccines (IPV) using attenuated poliovirus Sabin strains. Previously, we introduced a novel PER.C6® cell culture platform, which allows for high yield production of an affordable trivalent Sabin IPV vaccine.MethodsImmunogenicity and safety of this novel PER.C6®-based Sabin-IPV (sIPV) was assessed in rats and non-human primates (NHPs). NHPs received one of four different dose dilutions vaccine according to current human schedule (three prime-immunizations and one boost immunization). For comparison, NHPs received commercially available reference Salk IPV or sIPV.ResultsDose-dependent immunogenicity and good tolerability was observed for the PER.C6®-based sIPV formulations in rats and NHPs. In NHPs, the lowest tested dose that induced anti-Sabin virus-neutralizing antibody titers that were non-inferior to commercial sIPV after three immunizations was 5-7.5-25 D-antigen units for type 1, 2 and 3 respectively.DiscussionPER.C6®-based sIPV induced comparable immunogenicity to commercial Salk IPV and sIPV vaccines in NHPs. Together with the absence of any preclinical safety signals, these data warrant further testing in clinical trials. sIPV produced on the PER.C6® cell platform could be one solution to the need for an affordable and immunogenic IPV to achieve and maintain global polio eradication.     

Safety and immunogenicity of a primary series of Sabin-IPV with and without aluminum hydroxide in infants

Background

An inactivated poliovirus vaccine (IPV) based on attenuated poliovirus strains (Sabin-1, -2 and -3) was developed for technology transfer to manufacturers in low- and middle-income countries in the context of the global polio eradication initiative.

Method

Safety and immunogenicity of Sabin-IPV (sIPV) was evaluated in a double-blind, randomized, controlled, dose-escalation trial in the target population. Healthy infants (n = 20/group) aged 56–63 days, received a primary series of three intramuscular injections with low-, middle- or high-dose sIPV with or without aluminum hydroxide or with the conventional IPV based on wild poliovirus strains (wIPV). Virus-neutralizing titers against both Sabin and wild poliovirus strains were determined before and 28 days after three vaccinations.

Results

The incidence of local and systemic reactions was comparable with the wIPV. Seroconversion rates after three vaccinations were 100% for type 2 and type 3 polioviruses (both Sabin and wild strains) and 95–100% for type 1 polioviruses. Median titers were high in all groups. Titers were well above the log₂(titer) correlated with protection (=3) for all groups. Median titers for Sabin-2 were 9.3 (range 6.8–11.5) in the low-dose sIPV group, 9.2 (range 6.8–10.2) in the low-dose adjuvanted sIPV group and 9.8 (range 5.5–15.0) in the wIPV group, Median titers against MEF-1 (wild poliovirus type 2) were 8.2 (range 4.8–10.8) in the low-dose sIPV group, 7.3 (range 4.5–10.2) in the low-dose adjuvanted Sabin-IPV group and 10.3 (range 8.5–17.0) in the wIPV group. For all poliovirus types the median titers increased with increasing dose levels.

Conclusion

sIPV and sIPV adjuvanted with aluminum hydroxide were immunogenic and safe at all dose levels, and comparable with the wIPV.EudraCTnr: 2011-003792-11, NCT01709071.     

Immunogenicity and safety of the inactivated poliomyelitis vaccine made from Sabin strains in a phase IV clinical trial for the vaccination of a large population

As a recently launched novel vaccine used as one of the vaccines for the final eradication of polios worldwide, complete data on the consistency and immunogenicity characteristics of the inactivated poliomyelitis vaccine made from the Sabin strain (sIPV) and its safety in large-scale populations are required to support the future use of this vaccine worldwide. A phase IV clinical trial was conducted to perform an immunogenicity evaluation of lot-to-lot consistency of three commercial batches of sIPV in 1200 infants and to investigate the vaccine’s safety on a large-scale in 20,019 infants for active monitoring and 29,683 infants for passive monitoring through the Adverse Event Following Immunization (AEFI) reporting system in China. In the immunogenicity evaluation, the average seroconversion rates for type I, type II and type III of the three groups were 99.83%, 98.93% and 99.44%, respectively. No differences in the seroconversion rate and the GMT ratios were noted in the pair-to-pair comparisons. In the large-scale safety evaluation, most adverse reactions occurred 0–30 days after the first doses, and the common local and systemic reactions were similar to those in the phase III clinical trial, with low incidence in both activated and passive monitoring. In conclusion, sIPV exhibits good lot-to-lot consistency and safety in large-scale populations; thus, it is qualified to serve as one of the vaccines for use in eradicating all wild and vaccine-derived polioviruses worldwide in the near future.Clinic Trial Registration. NCT04224519 and NCT04220515.     

Sabin株脊髓灰质炎灭活疫苗与不同剂型Ⅰ型Ⅲ型脊髓灰质炎减毒活疫苗序贯接种的抗体滴度比较

目的  比较Sabin株脊髓灰质炎灭活疫苗(Sabin strain-based inactivated poliovirus vaccine,sIPV)与不同剂型Ⅰ型Ⅲ型脊髓灰质炎减毒活疫苗(bivalent types Ⅰ and Ⅲ oral poliovirus vaccine,bOPV)序贯接种的抗体滴度。 方法  选择柳州400名2月龄婴儿,按1：1：1：1随机分为sIPV+2bOPV糖丸、sIPV+2bOPV液体、2sIPV+bOPV糖丸、2sIPV+bOPV液体4组,按0 d、28 d、56 d免疫程序接种疫苗。检测免前、免后28 d脊灰中和抗体滴度。 结果  序贯程序相同时,1剂sIPV糖丸组与液体组免后Ⅰ型抗体滴度分布差异有统计学意义(Z=-2.589,P=0.010),Ⅱ型(Z=-0.331,P=0.741)和Ⅲ型(Z=-1.556,P=0.120)差异均无统计学意义(均有P>0.05);2剂sIPV糖丸组与液体组免后Ⅰ型(Z=-1.249,P=0.212)、Ⅱ型(Z=-1.658,P=0.097)和Ⅲ型(Z=-1.436,P=0.151)抗体滴度分布差异均无统计学意义(均有P>0.05)。剂型相同时,2剂sIPV组与1剂sIPV组各型抗体滴度分布差异均有统计学意义(均有P < 0.05)(1剂sIPV液体组与2剂sIPV液体组：Ⅰ型：Z=-2.766,P=0.006;Ⅱ型：Z=-9.137,P < 0.001;Ⅲ型：Z=-5.529,P < 0.001。1剂sIPV糖丸组与2剂sIPV糖丸组：Ⅰ型：Z=-3.748,P < 0.001;Ⅱ型：Z=-7.660,P < 0.001;Ⅲ型：Z=-6.030,P < 0.001)。 结论  bOPV糖丸和液体剂型免疫效果相似,各地接种部门应结合疫苗效果、接种对象特征和人口密度,选择适宜剂型接种;如sIPV供应充足,建议优先选择2剂sIPV序贯程序完成脊灰基础免疫接种。     

Evaluation of the genetic stability of Sabin strains and the consistency of inactivated poliomyelitis vaccine made from Sabin strains using direct deep-sequencing

Inactivated poliomyelitis vaccine made from Sabin strains (sIPV) has been encouraged to introduce in the “Global Polio Eradication & Endgame Strategic Plan” and increasingly used worldwide. Attenuated Sabin strains used in manufacture of oral poliovirus vaccine (OPV) and sIPV may regain full or partial neurovirulence during growth in vaccine recipients and the vaccine manufacturing processes. Ensuring the molecular consistency of sIPV batches and that no mutation accumulates beyond the level present in past batches are important for quality control of vaccine manufacture process. Direct deep-sequencing allows the construction of a library of virus RNA and the detection of genetic mutations throughout the viral genome. In the present study, direct deep-sequencing was conducted to detect molecular mutations in virus passages, multiple sIPV monovalent lots, and virus monovalent lots from different polio type III strains. The results indicated that direct deep-sequencing can be used to identify and quantify small amounts of mutant viruses in vaccine preparations, trace the source of a specific virus seed, and monitor the batch-to-batch consistency of vaccines, suggesting that this technique could be suitable for the quality control and consistency monitoring of sIPV production.     

延安市消灭脊髓灰质炎的策略与效果分析

目的评价延安市消灭脊髓灰质炎工作的策略与效果,为保证消灭脊灰提供依据。方法描述1956年以来脊髓灰质炎的发病状况和流行特征,分析不同时期该病防制策略。结果随着脊灰疫苗的广泛使用,以及消灭脊灰活动的深入开展,延安市脊灰发病得到有效控制,发病率逐年下降,自1991年以来,已连续14年无确诊病例发生;防治对策主要采取常规免疫、强化免疫与扫荡免疫相结合,病例报告与主动监测相结合。结论延安市已阻断脊灰野病毒的传播;脊灰疫情控制后,免疫策略应作相应调整,以控制疫苗相关病例的发生。     

Effects of the thermal denaturation of Sabin-derived inactivated polio vaccines on the D-antigenicity and the immunogenicity in rats

The efficacy of a Sabin-derived inactivated polio vaccine (sIPV) can be evaluated by measuring the immunogenicity and the contents of D-antigens, which induce the neutralizing antibodies. The immunogenic potency test in rats was done as a national assay in Japan. The two manufacturers of sIPV in Japan have performed both assays since development, and there is no clear discrepancy between the results obtained in the two assays. To further know the relationship between the two assays, we analyzed the effects of the heat treatment of sIPV on the D-antigenicity and the immunogenicity. We observed that the marginal D-antigen that remained after the thermal treatment was capable of inducing relatively high neutralizing antibodies in rats. This indicates that the measurement of the D-antigen contents as part of the quality control of sIPV is more sensitive and appropriate to detect denatured vaccines.     

Safety and immunogenicity of experimental stand-alone trivalent,inactivated Sabin-strain polio vaccine formulations in healthy infants: A randomized,observer-blind,controlled phase 1/2 trial

BackgroundTo increase the global supply of affordable IPV vaccine, preferably using Sabin viruses to comply with GAPIII requirements, Takeda has assessed three dosages of a stand-alone sIPV.MethodsIn this phase I/II study two cohorts of 40 adults and 60 toddlers, respectively, were initially assessed for safety after receiving high-dosage sIPV compared with placebo (adults) or Salk IPV (toddlers). A cohort of 240 infants was then enrolled and randomized (1:1:1:1) to receive low-, medium- or high-dosage sIPV, or a reference Salk IPV in a three-dose primary schedule at 6, 10 and 14 weeks of age. Parents completed safety diaries for 4 weeks after each dose, and immunogenicity was measured as neutralization antibody titers at baseline and four weeks after vaccination.ResultsAll vaccinations were generally well-tolerated and sIPV had a comparable safety profile to the control arm in adults or the reference Salk IPV vaccine in toddlers and infants. Infants displayed dosage-dependent immune responses to sIPV when assayed using Sabin strains, which were equivalent to the reference IPV in the high-dosage sIPV group for serotypes 1 and 2, but not for Sabin and Salk serotype 3. Seroconversion rates (SCR) of the low- and medium-dosage groups were significantly lower than the Salk IPV group for both Sabin and Salk serotypes 1 and type 2 (p < 0.05), with no significant differences for Salk or Sabin serotypes 3. Responses to sIPV, particularly to Sabin types 1 and 2, were higher in initially seronegative infants, indicating possible interference by maternally-derived antibodies.ConclusionsA novel stand-alone Sabin-based IPV vaccine was well tolerated with an acceptable safety profile, but less immunogenic than reference Salk IPV at 6, 10 and 14 weeks of age for Salk serotypes 1 and 2, with apparent interference by maternal antibodies. Additional preclinical assessments will be made before any further clinical development.